Novel hemopoietin receptor protein, nr10

ABSTRACT

The inventors succeeded in isolating a novel hemopoietin receptor gene (NR10) using a sequence predicted from the extracted motif conserved in the amino acid sequences of known hemopoietin receptors. It was expected that two forms of NR10 exists, a transmembrane type and soluble form. Expression of the former type was detected in tissues containing hematopoietic cells. Thus, NR10 is a novel hemopoietin receptor molecule implicated in the regulation of the immune system and hematopoiesis in vivo. These novel receptors are useful in screening for novel hematopoietic factors capable of functionally binding to the receptor, or developing medicines to treat diseases related with the immune system or hematopoietic system.

This application is a continuation claiming priority to U.S. applicationSer. No. 11/980,117 and U.S. application Ser. No. 11/980,135, both filedOct. 30, 2007, and each being a divisional application of U.S.application Ser. No. 10/006,265, filed Dec. 3, 2001, which is acontinuation-in-part of PCT/JP00/03556, filed Jun. 1, 2000, which claimspriority to Japanese Patent Application No. 11-155797, filed Jun. 2,1999 and Japanese Patent Application No. 11-217797, filed Jul. 30, 1999.Each of the prior U.S. applications is herein incorporated by reference.

TECHNICAL FIELD

The present invention relates to novel hemopoietin receptor proteins,and genes encoding them, as well as methods for producing and using thesame.

BACKGROUND

A large number of cytokines are known as humoral factors that areinvolved in the proliferation/differentiation of various cells and theactivation of functions of differentiated mature cells as well as celldeath. There are specific receptors for these cytokines, which arecategorized into several families based on their structural similarities(Hilton D. J., in “Guidebook to Cytokines and Their Receptors” edited byNicola N. A. (A Sambrook & Tooze Publication at Oxford UniversityPress), p 8-16, 1994).

On the other hand, as compared to the similarities of their receptors,the homology of the primary-structure among cytokines is quite low. Nosignificant amino acid homology has been observed, even among cytokinemembers that belong to the same receptor family. This explains thefunctional specificity of respective cytokines, as well as thesimilarities among cellular reactions induced by each cytokine.

Representative examples of the above-mentioned receptor families are thetyrosine kinase receptor family, the hemopoietin receptor family, thetumor necrosis factor (TNF) receptor family, and the transforming growthfactor β (TGF β) receptor family. Different signal transduction pathwayshave been reported to be involved in each of these families. Among thesereceptor families, many receptors of the hemopoietin receptor family inparticular are expressed in blood cells or immunocytes, and theirligands, cytokines, are often termed as hemopoietic factors orinterleukins. Some of these hemopoietic factors or interleukins existwithin blood and are thought to be involved in systemic humoralregulation of hemopoietic or immune functions.

This contrasts with the belief that cytokines belonging to otherfamilies are often involved in only topical regulations. Some of thesehemopoietins can be taken as hormone-like factors, and representativepeptide hormones, such as the growth hormone, prolactin, or leptinreceptors, also belong to the hemopoietin receptor family. Because ofthese hormone-like systemic regulatory features, it is anticipated thatadministration of these hemopoietins can be applied to the treatment ofvarious diseases.

Among the large number of cytokines, those that are presently beingclinically applied include erythropoietin, G-CSF, GM-CSF, and IL-2.Combined with IL-11, LIF, and IL-12 that are currently underconsideration for clinical trials, and the above-mentioned peptidehormones, such as growth hormone and prolactin, it can be envisaged thatby searching novel cytokines that bind to hemopoietin receptors amongthe above-mentioned various receptor families, it is possible to find acytokine that can be clinically applied with a higher efficiency.

As mentioned above, cytokine receptors share structural similaritiesamong the family members. Using these similarities, many investigationsare aimed at finding novel receptors. In particular, many receptors ofthe tyrosine kinase receptor family have already been cloned, using itshighly conserved sequence at the catalytic site (Matthews et al., Cell,65:1143-52, 1991). In comparison, hemopoietin receptors do not have atyrosine kinase-like enzyme activity domain in their cytoplasmicregions, and their signal transductions are known to be mediated throughassociations with other tyrosine kinase proteins existing freely in thecytoplasm.

Though the sites on receptors binding with these cytoplasmic tyrosinekinases (JAK kinases) are conserved among family members, the homologyis not very high (Murakami et al., Proc. Natl. Acad. Sci. USA,88:11349-11353, 1991). Actually, the sequence that best characterizesthese hemopoietin receptors exists in the extracellular region. Inparticular, a five amino acid motif, Trp-Ser-Xaa-Trp-Ser (SEQ ID NO:22),(wherein “Xaa” is an arbitrary amino acid), is conserved in almost allof the hemopoietin receptors. Therefore, novel receptors may be obtainedby searching for novel family members using this sequence. In fact,these approaches have already led to the identification of the IL-11receptor (Robb et al., J. Biol. Chem., 271:13754-13761, 1996), theleptin receptor (Gainsford et al., Proc. Natl. Acad. Sci. USA,93:14564-8, 1996), and the IL-13 receptor (Hilton et al., Proc. Natl.Acad. Sci. USA, 93:497-501, 1996).

SUMMARY

The present invention provides novel hemopoietin receptor proteins, andDNA encoding these proteins. The present invention also provides avector into which the DNA has been inserted, a transformant harboringthe DNA, and a method for producing recombinant proteins using thetransformant. The present invention also provides methods of screeningfor compounds that bind to the protein.

Initially, the inventors attempted to find a novel receptor usingoligonucleotides encoding the Trp-Ser-Xaa-Trp-Ser (SEQ ID NO:22) motif(WS motif), as the probe by plaque hybridization, RT-PCR method, and soon. However, it was extremely difficult to strictly select only those towhich all 15 nucleotides that encode the motif would completelyhybridize under the usual hybridization conditions, because theoligonucleotide tggag(t/c)nnntggag(t/c) wherein “n” is an arbitrarynucleotide (SEQ ID NO:21) encoding the motif was short, having just 15nucleotides, and had a high g/c content. Additionally, similar sequencesare contained within cDNA encoding proteins other than hemopoietinreceptors, starting with various collagens that are thought to be widelydistributed and also have high expression amounts, which makes thescreening by the above-mentioned plaque hybridization and RT-PCRextremely inefficient.

To solve these problems, the inventors searched for additional motifs,other than the site of the above-mentioned WS motif, that are conservedin the hemopoietin receptor family. As a result, a residue, eithertyrosine or histidine, located 13 to 27 amino acids upstream of the WSmotif in the extracellular domain was found to be highly conserved inthe receptor family. Furthermore, additional search for consensussequences that are frequently found in the 6 amino acids from the aboveTyr/His toward the C-terminus led to the identification of the followingconsensus sequence:(Tyr/His)-Xaa-(Hydrophobic/Ala)-(Gln/Arg)-Hydrophobic-Arg (hereinafter,abbreviated as the YR motif). However, this YR motif is not exactly aperfect consensus sequence, and the combination of the nucleotidesequence that encodes the motif is very complicated. Therefore, it ispractically impossible to synthesize and provide oligonucleotides thatencode all of the amino acid sequences as probes for hybridization,which is a practical method for screening, or as primers aimed forRT-PCR.

Accordingly, the inventors looked for other approaches to practicallysearch for novel members of the hemopoietin receptor family using theabove two motifs as probes, and determined that it would be appropriateto perform a database search on the computer using partial amino acidsequences of known hemopoietin receptors, including both motifs as thequery. The inventors repeated TBlastN searches on the gss and htgsdatabase in GenBank, using partial amino acid sequences from multipleknown hemopoietin receptors as the query. As a result, many falsepositive clones were obtained in all cases. However, by then comparingthe amino acid sequence deduced from the nucleotide sequence proximal tothe probe of the above clones with the sequence of known hemopoietinreceptors, the inventors were able to select genes that encode membersof the receptor family. From these results, the inventors identified asingle clone containing a human genomic sequence which was suspected toencode a novel hemopoietin receptor and named it NR10.

The above nucleotide sequence was used to design specificoligonucleotide primers. The primers were used to perform 5′- and3′-RACE using cDNA libraries from human fetal hepatocytes and humanplacenta as the template. As a result, a full-length cDNA, NR10.1,encoding a transmembrane receptor of 652 amino acids was isolated, andthe whole nucleotide sequence was determined. At the same time, a cDNAclone, NR10.2, presumed to be a splice variant of NR10, was alsosuccessfully isolated from the 3′-RACE product. Based on the determinednucleotide sequence, NR10.2 was suggested to encode a solublereceptor-like protein of 252 amino acids. It was revealed that thecysteine residues, proline-rich motif, and WSXWS (SEQ ID NO:22) motif,in the extracellular domain that is conserved among the receptor familymembers, the box1 motif in the intracellular domain that is implicatedin signal transduction, and so on were well conserved in the primarystructure of NR10.1. Therefore, NR10.1 was considered to encode atypical hemopoietin receptor.

Subsequently, RT-PCR was performed using mRNA prepared from varioushuman organs and primer sets specific to NR10.1 and NR10.2,respectively, to search for tissues expressing the respective genes andto examine their distribution and expression pattern in human tissues.The products of RT-PCR were subjected to Southern blotting using cDNAfragments specific to NR10.1 and NR10.2, respectively, in order todiscard the possibility of non-specific amplification and to quantifythe amount of the products. The results indicated that the NR10.2 geneis constitutively expressed in all tissues examined at a constant level.In contrast, the expression of NR10.1 was detected in restricted organsand tissues: in particular, strong expression was detected in adultheart, placenta, testis, thymus, and peripheral leukocytes, and weakexpression was detected in spleen, bone marrow, prostate, ovary,pancreas, and lung.

The inventors also performed PCR cloning to isolate the full-length openreading frame (ORF) of NR10.1, and by chance, isolated another cDNAclone, dubbed NR10.3, containing a nucleotide sequence in which a singlenucleotide is missing from the sequence of NR10.1 and encoding atransmembrane type receptor protein of 662 amino acids. NR10.3 wasconsidered to possess similar functions as NR10.1 from the closelyrelated structures.

Based on the above features, NR10 is presumed to be a novel hemopoietinreceptor molecule related to the regulation of the immune system orhematopoiesis in vivo. The gene encoding NR10 will be extremely usefulin screening for novel hematopoietic factors that can functionally bindto the receptor protein.

Consequently, this invention relates to novel hemopoietin receptors andgenes encoding the receptors, as well as a method for producing andusing the same. More specifically, the present invention provides thefollowing:

(1) a DNA selected from the group consisting of:

-   -   (a) a DNA encoding a protein consisting of the amino acid        sequence of any of SEQ ID NOs:2, 4, and 17;    -   (b) a DNA comprising the coding region of the nucleotide        sequence of any of SEQ ID NOs:1, 3, and 16;    -   (c) a DNA encoding a protein consisting of the amino acid        sequence of any of SEQ ID NOs:2, 4, and 17, in which one or more        amino acids are modified by deletion, addition and/or        substitution by another amino acid, wherein said protein is        functionally equivalent to the protein consisting of the amino        acid sequence of any of SEQ ID NOs:2, 4, and 17; and    -   (d) a DNA hybridizing under stringent conditions with a DNA        consisting of the nucleotide sequence of any of SEQ ID NOs:1, 3,        and 16, and encoding a protein that is functionally equivalent        to the protein consisting of the amino acid sequence of any of        SEQ ID NOs:2, 4, and 17;

(2) a DNA encoding a partial peptide of a protein consisting of theamino acid sequence of any of SEQ ID NOs:2, 4, and 17;

(3) a vector into which the DNA described in (1) or (2) is inserted;

(4) a transformant harboring the DNA described in (1) or (2) in anexpressible manner;

(5) a protein or peptide that is encoded by the DNA described in (1) or(2);

(6) a method for producing the protein or peptide of (5), comprising thesteps of: culturing the transformant of (4), and recovering theexpressed protein from said transformant or the culture supernatant;

(7) a method of screening for a compound that binds to the protein of(5), comprising the steps of:

-   -   (a) contacting a sample with the protein of (5) or partial        peptide thereof;

(b) detecting the binding activity of the sample with the protein of (5)or partial peptide thereof; and

-   -   (c) selecting the compound that binds to the protein of (5) or        partial peptide thereof;

(8) an antibody binding to the protein of (5);

(9) a method for detecting or measuring the protein of (5), comprisingthe steps of: exposing the antibody of (8) to a sample expected tocontain the protein of (5), and detecting or measuring the production ofthe immune complex between said antibody and said protein; and

(10) a polynucleotide complementary to either a DNA that comprises thenucleotide sequence of any of SEQ ID NOs:1, 3, and 16 or itscomplementary strand, wherein the polynucleotide comprises at least 15nucleotides.

This invention provides a novel hemopoietin receptor NR10. According tothe result of the database search on GenBank, 5′- and 3′-RACE analysis,the inventors finally succeeded in the identification and isolation of anovel hemopoietin receptor gene NR10. It was found that at least twosplice variants are transcribed from NR10. One of these variants, thecDNA clone NR10.1, encodes a transmembrane receptor protein, and theother, NR10.2, encodes a soluble receptor-like protein of 252 aminoacids. Furthermore, the inventors performed PCR cloning in order toisolate the full length ORF of the NR10.1 cDNA, and by chance, succeededin isolating another cDNA clone, named NR10.3, containing a full lengthORF encoding a transmembrane type receptor protein of 662 amino acids.

The nucleotide sequence of the NR10.1 cDNA and the amino acid sequenceof the protein encoded by the cDNA are shown in SEQ ID NOs:1 and 2,respectively. The nucleotide sequence of the NR10.2 cDNA and the aminoacid sequence of the protein encoded by the cDNA are shown in SEQ IDNOs:3 and 4, respectively. The nucleotide sequence of the NR10.3 cDNAand the amino acid sequence of a protein encoded by the cDNA are shownin SEQ ID NOs:16 and 17, respectively.

The NR10.3 cDNA clone has a single nucleotide deletion in the adeninecluster, located proximally to the stop codon, as compared with theNR10.1 clone, which results in a frame shift from that position leadingto a different open reading frame. Thus, the difference between the twoclones is not caused because they are transcription products of splicevariants. Except for the deletion of one nucleotide, NR10.1 and NR10.3cDNA clones share an identical sequence. Meanwhile, their extracellulardomains are encoded by a completely identical sequence and, thus, havean identical tertiary structure, and thereby, are considered torecognize the same specific ligand. Furthermore, their intracellulardomains share the Box1 motif (Pro-Xaa-Pro sequence following severalbasic residues and multiple hydrophobic residues) located immediatelyafter the transmembrane domain, and are presumed to bind to the JAKkinase. Therefore, it is predicted that the proteins encoded by the twoclones are functionally equivalent.

RT-PCR analysis using mRNA from various human organs revealed that theNR10.2 gene is constitutively expressed in all examined tissues at aconstant level. In contrast, expression of the NR10.1 gene was detectedin restricted tissues and organs: in particular, strong expression inadult heart, placenta, testis, thymus, and peripheral leukocytes, andweak expression in spleen, bone marrow, prostate, ovary, pancreas, andlung. Thus, it was presumed that NR10.1 encodes a novel hematopoieticfactor receptor.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Accordingly, the invention includes a polypeptide having a sequenceshown as SEQ ID NO:2, 4 or 17. The invention also includes apolypeptide, or fragment thereof, that differs from the correspondingsequence shown as SEQ ID NO:2, 4 or 17. The differences are, preferably,differences or changes at a non-essential residue or a conservativesubstitution. In one embodiment, the polypeptide includes an amino acidsequence at least about 60% identical to a sequence shown as SEQ IDNO:2, 4 or 17, or a fragment thereof. Preferably, the polypeptide is atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more identical toSEQ ID NO:2, 4 or 17 and has at least one hemopoietin receptor functionor activity described herein, e.g., the polypeptide binds to ahematopoietin factor. Preferred polypeptide fragments of the inventionare at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, ormore, of the length of the sequence shown as SEQ ID NO:2, 4 or 17 andhave at least one hemopoietin receptor function or activity describedherein. Or alternatively, the fragment can be merely an immunogenicfragment.

The above NR10 proteins may be useful for medical application. SinceNR10.1 is expressed in thymus, peripheral leukocytes, and spleen, itcould be a receptor for an unknown hematopoietic factor. Therefore, NR10proteins are a useful tool in the identification of the unknownhematopoietic factor. They may also be used to screen a peptide libraryor synthetic chemical compounds in order to isolate or identify agonistsor antagonists that can functionally bind to the NR10 molecule.Moreover, clinical application is expected of novel molecules binding tothe NR10 molecule and specific antibodies that can limit the function ofthe NR10 molecule to regulate the immune response or hematopoiesis invivo, by searching such molecules and antibodies.

NR10 is expected to be expressed in a restricted population of cells inthe hematopoietic tissues, and thus, anti-NR10 antibodies are useful forthe isolation of such cell populations. The isolated cell populationsmay be used in cell transplantation. Furthermore, it is expected thatthe anti-NR10 antibody may be used for the diagnosis or treatment ofdiseases, such as leukemia.

On the other hand, the soluble proteins comprising the extracellulardomain of NR10 protein and the splice variant of NR10, NR10.2, may beused as a decoy-type receptor to inhibit the NR10 ligand. They may beuseful for treatment of diseases in which NR10 is implicated, such asleukemia.

This invention includes proteins that are functionally equivalent to theNR10 protein. For instance, homologues of human NR10 protein in otherspecies and mutants of human NR10 protein are included. Herein, the term“functionally equivalent” refers to proteins having an equivalentbiological activity as compared to that of an NR10 protein. Suchbiological activity may include the protein activity as a membrane boundor soluble form hematopoietic factor receptor.

Methods of introducing mutations for preparing proteins that arefunctionally equivalent to another protein are well known to one skilledin the art. For example, one may use site-directed mutagenesis(Hashimoto-Goto et al., Gene, 152:271-275, 1995; Zoller et al., MethodsEnzymol., 100:468-500, 1983; Kramer et al., Nucl. Acids Res.,12:9441-9456, 1984; Kramer et al., Methods. Enzymol., 154:350-367, 1987;Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492, 1985; Kunkel, MethodsEnzymol., 85:2763-2766, 1988) and such in order to introduce anappropriate mutation into the amino acid sequence of human NR10 proteinand prepare a protein that is functionally equivalent to the protein.Mutations of amino acids may occur in nature as well. This inventionincludes proteins having the amino acid sequence of human NR10 proteinin which one or more amino acid residues are mutated, and wherein theproteins are functionally equivalent to human NR10 protein.

As a protein functionally equivalent to the NR10 protein of theinvention, the following can be specifically mentioned: one in which oneor two or more, preferably, two to 30, more preferably, two to ten aminoacids are deleted in any one of the amino acid sequences of SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:17; one in which one or two or more,preferably, two to 30, more preferably, two to ten amino acids have beenadded into any one of the amino acid sequences of SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:17; or one in which one or two or more, preferably,two to 30, more preferably, two to ten amino acids have been substitutedwith other amino acids in any one of the amino acid sequences of SEQ IDNO:2, SEQ ID NO:4 or SEQ ID NO:17.

As for the amino acid residue to be mutated, it is preferable that it bemutated into a different amino acid that allows the properties of theamino acid side-chain are conserved. Examples of properties of aminoacid side chains are the following: hydrophobic amino acids (A, I, L, M,F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S,T), and amino acids comprising the following side chains: an aliphaticside-chain (G, A, V, L, I, P); a hydroxyl group containing side-chain(S, T, Y); a sulfur atom containing side-chain (C, M); a carboxylic acidand amide containing side-chain (D, N, E, Q); a base-containingside-chain (R, K, H); and an aromatic-containing side-chain (H, F, Y, W)(The parenthetic letters indicate the one-letter codes of amino acids).

It is known that a protein may have an amino acid sequence of which ismodified by deletion, addition, and/or substitution by other amino acidsof one or more amino acid residues, yet still retain its biologicalactivity (Mark et al., Proc. Natl. Acad. Sci. USA, 81:5662-5666, 1984;Zoller et al., Nucl. Acids Res., 10:6487-6500, 1982; Wang et al.,Science, 224:1431-1433; Dalbadie-McFarland et al., Proc. Natl. Acad.Sci. USA, 79:6409-6413, 1982).

A fusion protein containing human NR10 protein is an example of aprotein in which one or more amino acid residues have been added to theamino acid sequence (SEQ ID NO:2, 4 or 17) of human NR10 protein. Afusion protein is made by fusing the human NR10 protein of the presentinvention with another peptide(s) or protein(s) and is included in thepresent invention. A fusion protein can be prepared by ligating a DNAencoding the human NR10 protein of the present invention with a DNAencoding another peptide(s) or protein(s) in frame, introducing theligated DNA into an expression vector, and expressing the fusion gene ina host. Methods known by one skilled in the art can be used forpreparing such a fusion gene. There is no restriction as to the otherpeptide(s) or protein(s) that is (are) fused to the protein of thisinvention.

Other peptide(s) to be fused with a protein of the present invention areknown peptides, for example, FLAG (Hopp et al., Biotechnology,6:1204-1210, 1988), 6×His constituting six histidine (His) residues,10×His, Influenza agglutinin (HA), human c-myc fragment, VSV-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, and soon. Other examples of peptides to be fused with the protein of thepresent invention are the glutathione-S-transferase (GST), Influenzaagglutinin (HA), immunoglobulin constant region, β-galactosidase,maltose-binding protein (MBP), etc.

Fusion proteins can be prepared by fusing commercially available DNAencoding these peptides or proteins with DNA encoding a protein of thepresent invention and expressing the fused DNA prepared.

The hybridization technique (Sambrook et al., Molecular Cloning 2nd ed.9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to oneskilled in the art as an alternative method for preparing a proteinfunctionally equivalent to a certain protein. More specifically, oneskilled in the art can utilize the general procedure to obtain a proteinfunctionally equivalent to a human NR10 protein by isolating DNA havinga high homology with the whole or part of the DNA (SEQ ID NO:1, 3 or 16)encoding the human NR10 protein. Thus, the present invention includessuch proteins, that are encoded by DNAs that hybridize with a DNAconsisting of a DNA encoding human NR10 protein or part thereof and thatare functionally equivalent to a human NR10 protein. For instance,homologues of human NR10 in other mammals (such as those of monkey,mouse, rabbit, and bovine) are included. In order to isolate a cDNA withhigh homology to a DNA encoding a human NR10 protein from animals, it ispreferable to use tissues such as heart, placenta, and testis.

Stringent hybridization conditions for isolating DNA encodingfunctionally equivalent proteins of human NR10 protein can be suitablyselected by one skilled in the art, and for example, low-stringentconditions can be given. Low-stringent conditions are, for example, 42°C., 2×SSC, and 0.1% SDS, and preferably, 50° C., 2×SSC, and 0.1% SDS.Highly stringent conditions are more preferable and include, forexample, 65° C., 2×SSC, and 0.1% SDS. Under these conditions, the higherthe temperature, the higher the homology of the obtained DNA will be.However, several factors other than temperature, such as saltconcentration, can influence the stringency of hybridization and oneskilled in the art can suitably select the factors to accomplish asimilar stringency.

In place of hybridization, the gene amplification method, for example,the polymerase chain reaction (PCR) method can be utilized to isolatethe object DNA using primers synthesized based on the sequenceinformation of the DNA encoding the human NR10 protein (SEQ ID NO:1, 3and 16).

Proteins that are functionally equivalent to human NR10 protein, encodedby DNA isolated through the above hybridization technique or by the geneamplification technique, normally have a high homology to the amino acidsequence of the human NR10 protein. The proteins of the presentinvention also include proteins that are functionally equivalent to thehuman NR10 protein, which also have a high homology with the proteincomprising any one of the amino acid sequences of SEQ ID NO:2, 4, or 17.High homology is defined normally as a homology of 70% or higher,favorably 80% or higher, more favorably 90% or higher, and mostfavorably 95% or higher. The homology of a protein can be determined bythe algorithm in “Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci.USA 80: 726-730 (1983).”

The amino acid sequence, molecular weight, isoelectric point, thepresence or absence of the sugar chain, and the form of a protein of thepresent invention may differ according to the producing cells, host, orpurification method described below. However, so long as the obtainedprotein has an equivalent function to a protein of the present invention(SEQ ID NO:2, 4 and 17), it is included in the present invention. Forexample, if a protein of the present invention is expressed inprokaryotic cells, such as E. coli, a methionine residue is added at theN-terminus of the amino acid sequence of the expressed protein. If aprotein of the present invention is expressed in eukaryotic cells, suchas mammalian cells, the N-terminal signal sequence is removed. Suchproteins are also included in the proteins of the present invention.

For example, as the result of analysis of the protein of the inventionbased on the method in “Von Heijne, G., Nucl. Acids Res., 14: 4683-4690(1986)”, it was presumed that the signal sequence is from the 1st Met tothe 32nd Ala in the amino acid sequence of SEQ ID NO:2, 4 and 17.Therefore, the present invention encompasses a protein comprising thesequence from the 33rd Ala to 652nd Asp in the amino acid sequence ofSEQ ID NO:2. Similarly, the present invention encompasses a proteincomprising the sequence from the 33rd Ala to 252nd Val in the amino acidsequence of SEQ ID NO:4. Similarly, the present invention encompasses aprotein comprising the sequence from the 33rd Ala to 662nd Ile in theamino acid sequence of SEQ ID NO:17.

A protein of the present invention can be prepared by methods known toone skilled in the art, as a recombinant protein, and also as a naturalprotein. A recombinant protein can be prepared by inserting a DNAencoding a protein of the present invention (for example, the DNAcomprising the nucleotide sequence of SEQ ID NO:1, 3 or 16) into asuitable expression vector, introducing the vector into a suitable hostcell, and collecting the protein from the resulting transformant. Afterobtaining the extract, recombinant protein can be purified and preparedby subjecting to chromatography, such as ion exchange chromatography,reverse phase chromatography, gel filtration, and such, or affinitychromatography, to which antibodies against the protein of the inventionare immobilized, or combining one or more of these columns.

Further, when a protein of the present invention is expressed withinhost cells (for example, animal cells and E. coli), as a fusion proteinwith glutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or nickel column.

After purifying the fusion protein, it is also possible to excluderegions other than the objective protein by cutting with thrombin,factor-Xa, and such, as required.

A natural protein may be isolated by methods known to one skilled in theart. For instance, extracts of tissue or cells expressing a protein ofthe invention may be reacted with an affinity column described below, towhich antibodies binding to the NR10 protein are attached, to isolatethe natural protein. Polyclonal or monoclonal antibodies may be used.

This invention also includes partial peptides of the proteins of theinvention. The peptide consisting of the amino acid sequence specific toa protein of the invention are composed of at least 7 amino acids,favorably more than 8 amino acids, and more favorably more than 9 aminoacids. The partial peptides may be useful for preparing antibodiesagainst a protein of the invention, or screening compounds binding to aprotein of the present invention, or screening activators or inhibitorsof a protein of the present invention. Alternatively, it may be used asan antagonist for the ligand of a protein of the invention. A partialpeptide of the present invention is, for example, a partial peptidehaving the active center of the protein consisting of any one of theamino acid sequences of SEQ ID NO:2, 4, or 17. Additionally, the partialpeptides may contain one or more regions of the hydrophilic regions orhydrophobic regions presumed by hydrophobicity plot analysis. Thesepartial peptides may contain the whole or a part of a hydrophilicregion, or may contain the whole or a part of a hydrophobic region.Moreover, for example, soluble proteins and proteins comprisingextracellular regions of a protein of the invention are also encompassedin the present invention.

The partial peptides of the invention may be produced by geneticengineering techniques, well-known peptide synthesizing methods, or byexcising a protein of the invention with a suitable peptidase. Thesolid-phase synthesizing method and the liquid-phase synthesizing methodmay be used as peptide synthesizing methods.

Another objective of this invention is to provide DNA encoding a proteinof the invention. The DNA may be useful for producing the above proteinsof the invention in vivo and in vitro. Furthermore, for example, it isalso possible to use the DNA for application to gene therapy and such ofdiseases arising from abnormalities of the gene encoding the protein ofthe present invention. The DNA may be provided in any form as long as itencodes a protein of the invention. Thus, the DNA may be a cDNAsynthesized from mRNA, genomic DNA, or chemically synthesized DNA.Furthermore, a DNA comprising any nucleotide sequence based on thedegeneracy of genetic code may be included as long as it encodes aprotein of the present invention.

As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

Accordingly, in one aspect, the invention provides an isolated orpurified nucleic acid molecule that encodes a polypeptide describedherein or a fragment thereof. Preferably, the isolated nucleic acidmolecule includes a nucleotide sequence that is at least 60% identicalto the nucleotide sequence shown in SEQ ID NO:1, 3 or 16. Morepreferably, the isolated nucleic acid molecule is at least 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, identical to the nucleotide sequence shown in SEQ ID NO:1, 3 or16. In the case of an isolated nucleic acid molecule which is longerthan or equivalent in length to the reference sequence, e.g., SEQ IDNO:1, 3 or 16, the comparison is made with the full length of thereference sequence. Where the isolated nucleic acid molecule is shorterthat the reference sequence, e.g., shorter than SEQ ID NO:1, 3 or 16,the comparison is made to a segment of the reference sequence of thesame length (excluding any loop required by the homology calculation).

As used herein, “% identity” of two amino acid sequences, or of twonucleic acid sequences, is determined using the algorithm of Karlin andAltschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990), modified asin Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993).Such an algorithm is incorporated into the NBLAST and)(BLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the)(BLASTprogram, score=50, wordlength=3. To obtain gapped alignment forcomparison purposes GappedBLAST is utilized as described in Altschul etal (Nucl. Acids Res., 25:3389-3402, 1997). When utilizing BLAST andGappedBLAST programs the default parameters of the respective programs(e.g., XBLAST and NBLAST) are used to obtain nucleotide sequenceshomologous to a nucleic acid molecule of the invention.

The DNA of the invention can be prepared by any method known to oneskilled in the art. For instance, the DNA may be prepared byconstructing a cDNA library from cells expressing the protein, andperforming hybridization using a partial sequence of the DNA of theinvention (SEQ ID NO:1 or 3, for instance) as a probe. A cDNA librarymay be constructed according to the method described in the literature(Sambrook et al., Molecular Cloning, Cold Spring Harbor LaboratoryPress, 1989), or a commercial DNA library may be used. Alternatively,the DNA may be prepared by obtaining RNA from a cell expressing aprotein of the present invention, synthesizing oligo DNA based on thesequence of the present DNA (SEQ ID NO:1, 3 or 16, for instance),performing PCR using the synthesized DNA as the primer, and amplifyingthe cDNA encoding a protein of the present invention.

By determining the nucleotide sequence of the obtained cDNA, thetranslation region encoded by the cDNA can be determined, and the aminoacid sequence of the protein of the present invention can be obtained.Furthermore, genomic DNA can be isolated by screening genomic DNAlibraries using the obtained cDNA as a probe.

Specifically, this can be done as follows: First, mRNA is isolated fromcells, tissues, and organs (for example, ovary, testis, placenta, etc.)expressing a protein of the invention. To isolate the mRNA, at first,whole RNA is prepared using well-known methods, for example, guanidineultracentrifugation method (Chirgwin et al., Biochemistry, 18:5294-5299,1979), the AGPC method (Chomczynski et al., Anal. Biochem., 162:156-159,1987), and such, and mRNA from whole mRNA is purified using the mRNAPurification Kit (Pharmacia), etc. Alternatively, mRNA may be directlyprepared using the QuickPrep mRNA Purification Kit (Pharmacia).

cDNA is synthesized using reverse transcriptase from the obtained mRNA.cDNA can be synthesized by using the AMV Reverse TranscriptaseFirst-strand cDNA Synthesis Kit (SEIKAGAKU CORPORATION), etc.Additionally, cDNA synthesis and amplification may be also done by usingthe primer and such described herein following the 5′-RACE method(Frohman et al., Proc. Natl. Acad. Sci. USA, 85:8998-9002, 1988;Belyaysky et al., Nucl. Acids Res., 17:2919-2932, 1989) utilizing thepolymerase chain reaction (PCR) and the 5′-Ampli FINDER RACE KIT(Clontech).

The objective DNA fragment is prepared from the obtained PCR product andligated with the vector DNA. Thus, a recombination vector is created,introduced into E. coli, and such, and colonies are selected to preparethe desired recombination vector. The nucleotide sequence of theobjective DNA can be verified by known methods, for example, the dideoxynucleotide chain termination method.

With regards to the DNA of the invention, a sequence with higherexpression efficiency can be designed by considering the codon usagefrequency in the host used for the expression (Grantham et al., Nucl.Acids Res., 9:43-74, 1981). The DNA of the invention may also bemodified using commercially available kits and known methods.Modifications are given as, for example, digestion by restrictionenzymes, insertion of synthetic oligonucleotides and suitable DNAfragments, addition of linkers, insertion of a start codon (ATG) and/orstop codon (TAA, TGA, or TAG), and such.

Specifically, the DNA of the invention includes DNA consisting of thenucleotide sequence from the 523rd “A” to 2478th “C” of SEQ ID NO:1,523rd “A” to 1278th “G” of SEQ ID NO:3, or 11th “A” to 1996th “A” of SEQID NO:16.

The DNA of the present invention includes DNA that hybridize understringent conditions to the DNA consisting of any one of thenucleotides, wherein the DNA encodes a protein functionally equivalentto an above-mentioned protein of the present invention.

Stringent conditions can be suitably selected by one skilled in the art,and for example, low-stringent conditions can be given. Low-stringentconditions are, for example, 42° C., 2×SSC, and 0.1% SDS, and preferably50° C., 2×SSC, and 0.1% SDS. More preferable are highly stringentconditions which are, for example, 65° C., 2×SSC, and 0.1% SDS. Underthese conditions, the higher the temperature, the higher the homology ofthe obtained DNA will be. The above DNA hybridizing to the DNA with thesequences of SEQ ID NO:1, 3, and 16, is preferably a natural DNA such ascDNA and chromosomal DNA.

Moreover, the present invention provides a vector containing a DNA ofthe invention as an insert. The vector may be useful for maintaining theDNA in host cells or producing the protein of the invention.

If the host cell is E. coli (such as JM109, DH5α, HB101, and XL1Blue),any vector may be used as long as it contains the “ori” foramplification in E. coli that enables large-scale preparation, and aselection marker for transformants (for instance, a drug resistance genethat enables selection by a drug such as ampicillin, tetracycline,kanamycin, and chloramphenicol). For instance, series of the M13 vectorsand pUC vectors, pBR322, pBluescript, pCR-Script, and so on may be used.For the purpose of subcloning or excision of a cDNA, pGEM-T, pDIRECT,pT7, and such may be used as well. For producing the protein of theinvention, an expression vector is especially useful. For instance, ifthe protein is to be expressed in E. coli, the expression vector musthave such characteristics as above to be amplified in E. coli, and apromoter for efficient expression, such as the lacZ promoter (Ward etal., Nature, 341:544-546, 1989; FASEB J., 6:2422-2427, 1992), araBpromoter (Better et al., Science, 240:1041-1043, 1988), or T7 promoter.Such vector includes pGEX-5X-1 (Pharmacia), vectors in the QIAexpresssystem (QIAGEN), pEGFP, pET (BL21 expressing the T7 RNA polymerase isfavorably used as the host), and so on except those mentioned above.

The vector may contain a signal sequence for polypeptide secretion. ThepelB signal sequence (Lei et al., J. Bacteriol., 169:4379, 1987) may beused to produce the proteins in the periplasm of E. coli. Vectors may beintroduced into host cells, for example, by the calcium chloride methodor electroporation.

For example, the expression vector to prepare the protein of theinvention may be a mammal-derived expression vector (e.g., pcDNA3(Invitrogen), pEGF-BOS (Nucl. Acids. Res., 18:5322, 1990), pEF andpCDM8), an insect cell-derived expression vector (e.g., “Bac-to-BACbaculovirus expression system” (GIBCO BRL), pBacPAK8), a plant-derivedexpression vector (e.g., pMH1 and pMH2), an animal virus-derivedexpression vector (e.g., pHSV, pMV, and pAdexLcw), a retrovirus-derivedexpression vector (e.g., pZIpneo), an yeast-derived expression vector(e.g., “Pichia Expression Kit” (In vitrogen), pNV11 and SP-Q01), or aBacillus subtilis-derived expression vector (e.g., pPL608 and pKTH50),other than E. coli.

For the expression in animal cells, such as CHO, COS, and NIH3T3 cells,the expression vector must have a promoter such as SV40 promoter(Mulligan et al., Nature, 277:108, 1979), MMLV-LTR promoter, EF1αpromoter (Mizushima et al., Nucl. Acids Res., 18:5322, 1990), and CMVpromoter. More favorably, the vector may contain a marker for theselection of transfected cells (for instance, a drug resistance gene forselection by a drug such as neomycin and G418). Such vectors includepMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13, and so on.

Furthermore, in order to achieve stable gene expression andamplification of the copy number of genes in cell, CHO cells deficientin the metabolic pathway for nucleotide synthesis may be used. The CHOcell is transfected with an expression vector containing the DHFR genethat complements the deficiency (such as pCHOI), then the vector may beamplified by methotrexate (MTX) treatment. For transient geneexpression, COS cells containing a gene expressing the SV40 T-antigen onits chromosome may be used to transform with a vector containing theSV40 replication origin (such as pCD). Examples of replication originsto be used in the present invention include those derived frompolyomavirus, adenovirus, bovine papilomavirus (BPV), and such.Moreover, to amplify the gene copies in host cell lines, the expressionvector may include an aminoglycoside transferase (APH) gene, thymidinekinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase(Ecogpt) gene, dihydrofolate reductase (dhfr) gene, and such as aselective marker.

In vivo expression of the DNA of the invention may be performed byconstructing the DNA into an appropriate vector and transfecting theconstruct into the body using retrovirus, liposome, cationic liposome,adenovirus, and so on. It is possible to use such a construct to performgene therapy for diseases that arise from mutations in the NR10 gene.Examples of vectors used for this purpose include an adenovirus vector(such as pAdexlcw) and a retrovirus vector (such as pZIPneo), but arenot limited thereto. General manipulations, such as insertion of the DNAinto the vector, may be performed by using standard methods (MolecularCloning, 5.61-5.63). The vector may be administered to the patientthrough ex vivo or in vivo methods.

Another object of this invention is to provide a transformant thatcontains the DNA of the invention in a expressible manner. The host cellto insert the vector of the present invention is not limited in any way,and E. coli, a variety of animal cells, and so on may be used. Thetransformant may be used as a producing system for preparing orexpressing a protein of the invention. In vitro and in vivo productionsystems are known as production systems for producing proteins.Production systems using eukaryotic cells and prokaryotic cells may beused as the in vitro production systems.

When using eukaryotic cells, production systems using, for example,animal cells, plant cells, and fungal cells are available as hosts.Examples animal cells used include mammalian cells such as CHO (J. Exp.Med., 108:945, 1995), COS, 3T3, myeloma, baby hamster kidney (BHK),HeLa, Vero, amphibian cells such as Xenopus oocytes (Valle et al.,Nature, 291:338-340, 1981), insect cells such as sf9, sf21, or Tn5. AsCHO cells, especially DHFR gene-deficient CHO cell, dhfr-CHO (Proc.Natl. Acad. Sci. USA, 77:4216-4220, 1980), and CHO K-1 (Proc. Natl.Acad. Sci. USA, 60:1275, 1968) can be suitably used. For large-scalepreparation in animal cells, CHO cells may be favorably used. The vectormay be transfected into host cells using a variety of methods, such asthose using calcium phosphate, DEAE-dextran, or cationic liposome DOTAP(Boeringer Mannheim), as well as electroporation, lipofection, and soon.

Nicotiana tabacum-derived cells are well known as protein productionsystems in plant cells, and these can be callus cultured. As fungalcells, yeasts such as the Saccharomyces genus, for example,Saccharomyces cerevisiae; filamentous bacteria, such as Aspergillusgenus, for example, Aspergillus niger are known.

Bacterial cells may be used as prokaryotic production systems. Asbacterial cells, E. coli, for example, JM109, DH5α, HB101, and such, aswell as others like Bacillus subtilis are known.

Proteins can be obtained by transforming these cells with the objectiveDNA, and culturing the transformed cells in vitro according to knownmethods. For example, DMEM, MEM, RPMI1640, and IMDM can be used asculture media of animal cells. Occasionally, fetal calf serum (FCS) andsuch serum supplements may be added in the above media; alternatively, aserum-free culture medium may be used. The pH is preferably from about 6to 8. The culturing is usually performed at about 30° C. to 40° C., forabout 15 to 200 hr, and medium changes, aeration, and stirring is doneas necessary.

On the other hand, for example, production systems using animals andplants may be given as in vivo protein production systems. The objectiveDNA is introduced into the plant or animal, and the protein is producedwithin the plant or animal, and then, the protein is recovered. The term“host” as used in the present invention encompasses such animals andplants as well.

When using animals, mammalian and insect production systems can be used.As mammals, goats, pigs, sheep, mice, and bovine may be used (VickiGlaser, SPECTRUM Biotechnology Applications (1993)). Transgenic animalsmay also be used when using mammals.

For example, the objective DNA is prepared as a fusion gene with a geneencoding a protein intrinsically produced into milk, such as goat βcasein. Next, the DNA fragment containing the fusion gene is injectedinto goat's embryo, and this embryo is implanted in female goat. Theobjective protein can be collected from the milk of the transgenic goatsproduced from the goat that received the embryo, and descendantsthereof. To increase the amount of protein-containing milk produced fromthe transgenic goat, a suitable hormone/hormones may be given to thetransgenic goats (Ebert et al., Bio/Technology, 12:699-702, 1994).

Silk worms may be used as insects. When using silk worms, they areinfected with baculoviruses to which the DNA encoding objective proteinhas been inserted, and the desired protein can be obtained from the bodyfluids of the silk worm (Susumu et al., Nature, 315:592-594, 1985).

When using plants, for example, tobacco can be used. In the case oftobacco, the DNA encoding the objective protein is inserted into a plantexpression vector, for example, pMON 530, and this vector is introducedinto a bacterium such as Agrobacterium tumefaciens. This bacterium isinfected to tobacco, for example, Nicotiana tabacum, and it is able toobtain the desired polypeptide from the tobacco leaves (Julian et al.,Eur. J. Immunol., 24:131-138, 1994).

Thus-obtained protein of the invention is isolated from inside oroutside (medium, etc.) the host cell, and may be purified as asubstantially pure homogenous protein. The separation and purificationof the protein can be done using conventional separation andpurification methods used to purify proteins and are not limited to anyspecific method. For example, chromatography column, filtration,ultrafiltration, salting out, solvent precipitation, solvent extraction,distillation, immunoprecipitation, SDS-polyacrylamide gelelectrophoresis, isoelectric focusing, dialysis, recrystallization, andsuch may be suitably selected, or combined to separate/purify theprotein.

For example, affinity chromatography, ion exchange chromatography,hydrophobic chromatography, gel filtration, reversed-phasechromatography, adsorption chromatography, and such can be exemplifiedas chromatographies (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press, 1996). These chromatographiescan be performed by liquid chromatography, such as HPLC, FPLC, and thelike. The present invention encompasses proteins highly purified byusing such purification methods.

Proteins can be arbitrarily modified, or peptides may be partiallyexcised by treating the proteins with appropriate modification enzymesprior to or after the purification. Trypsin, chymotrypsin, lysylendopeptidase, protein kinase, glucosidase, and such are used as proteinmodification enzymes.

The present invention also provides antibodies binding to the protein ofthe invention. There is no particular restriction as to the form of theantibody of the present invention and the present invention includespolyclonal antibodies, as well as monoclonal antibodies. The antiserumobtained by immunizing animals such as rabbits with a protein of thepresent invention, as well polyclonal and monoclonal antibodies of allclasses, human antibodies, and humanized antibodies made by geneticengineering, are also included.

A protein of the invention that is used as a sensitizing antigen forobtaining antibodies is not restricted by the animal species from whichit is derived, but is preferably a protein derived from mammals, forexample, humans, mice, or rats, especially preferably from humans.Proteins of human origin can be obtained by using the nucleotidesequence or amino acid sequence disclosed herein.

Herein, an intact protein or its partial peptide may be used as theantigen for immunization. As partial peptides of the proteins, forexample, the amino (N) terminal fragment of the protein, and the carboxy(C) terminal fragment can be given. “Antibody” as used herein means anantibody that specifically reacts with the full-length or fragments ofthe protein.

A gene encoding a protein of the invention or a fragment thereof isinserted into a well-known expression vector, and by transforming thehost cells with the vector described herein, the objective protein or afragment thereof is obtained from inside or outside the host cell usingwell-known methods, and this protein can be used as the sensitizingantigen. Also, cells expressing the protein, cell lysates, or chemicallysynthesized protein of the invention may be also used as a sensitizingantigen.

The mammals that are immunized by the sensitizing antigen are notrestricted, but it is preferable to select animals by considering theadaptability with the parent cells used in cell fusion. Generally,animals belonging to the rodentia, lagomorpha, and Primate family areused.

Examples of animals belonging to rodentia that may be used include mice,rats, hamsters, and such. Examples of animals belonging to lagomorphathat may be used include, for example, rabbits. Examples of Primatesthat may be used include monkeys. Examples of monkeys to be used includethe infraorder catarrhini (Old World Monkeys), for example, cynomolgusmonkeys, rhesus monkeys, sacred baboons, chimpanzees, and such.

Well-known methods may be used to immunize animals with the sensitizingantigen. For example, the sensitizing antigen is generally injectedintraperitoneally or subcutaneously into mammals. Specifically, thesensitizing antigen is suitably diluted and suspended in physiologicalsaline or phosphate-buffered saline (PBS) and mixed with a suitableamount of general adjuvant if desired, for example, with Freund'scomplete adjuvant. Then, the solution is emulsified and injected intothe mammal. Thereafter, the sensitizing antigen suitably mixed withFreund's incomplete adjuvant is preferably given several times every 4to 21 days. A suitable carrier can also be used when immunizing ananimal with the sensitizing antigen. After the immunization, theelevation in the level of serum antibody is detected by usual methods.

Polyclonal antibodies against a protein of the invention can be obtainedas follows. After verifying that a desired serum antibody level has beenreached, blood is withdrawn from the mammal sensitized with the antigen.Serum is isolated from this blood using well-known methods. The serumcontaining the polyclonal antibody may be used as the polyclonalantibody, or according to needs, the polyclonal antibody-containingfraction may be further isolated from the serum. For instance, afraction of antibodies that specifically recognize the protein of theinvention may be prepared by using an affinity column to which theprotein is coupled. Then, the fraction may be further purified by usinga Protein A or Protein G column in order to prepare immunoglobulin G orimmunoglobulin M.

To obtain monoclonal antibodies, after verifying that the desired serumantibody level has been reached in the mammal sensitized with theabove-described antigen, immunocytes are taken from the mammal and usedfor cell fusion. For this purpose, splenocytes can be mentioned aspreferable immunocytes. As parent cells fused with the aboveimmunocytes, mammalian myeloma cells are preferably used. Morepreferably, myeloma cells that have acquired the feature, which can beused to distinguish fusion cells by agents, are used as the parent cell.

The cell fusion between the above immunocytes and myeloma cells can beconducted according to known methods, for example, the method ofMilstein et al. (Methods Enzymol., 73:3-46, 1981).

The hybridoma obtained from cell fusion is selected by culturing thecells in a standard selective culture medium, for example, HAT culturemedium (hypoxanthine, aminopterin, thymidine-containing culture medium).The culture in this HAT medium is continued for a period sufficientenough for cells (non-fusion cells) other than the objective hybridomato perish, usually from a few days to a few weeks. Next, the usuallimiting dilution method is carried out, and the hybridoma producing theobjective antibody is screened and cloned.

Other than the above method for obtaining hybridomas, by immunizing ananimal other than humans with the antigen, a hybridoma producing theobjective human antibodies having the activity to bind to proteins canbe obtained by the method of sensitizing human lymphocytes, for example,human lymphocytes infected with the EB virus, with proteins,protein-expressing cells, or lysates thereof in vitro, fusing thesensitized lymphocytes with myeloma cells derived from human, forexample U266, having a permanent cell division ability (UnexaminedPublished Japanese Patent Application (JP-A) No. Sho 63-17688).

The obtained monoclonal antibodies can be purified by, for example,ammonium sulfate precipitation, protein A or protein G column, DEAE ionexchange chromatography, an affinity column to which the protein of thepresent invention is coupled, and so on. The antibody may be useful forthe purification or detection of a protein of the invention. It may alsobe a candidate for an agonist or antagonist of the protein. Furthermore,it is possible to use it for the antibody treatment of diseases in whichthe protein is implicated. For in vivo administration (in such antibodytreatment), human antibodies or humanized antibodies may be favorablyused because of their reduced antigenicity.

For example, a human antibody against a protein can be obtained usinghybridomas made by fusing myeloma cells with antibody-producing cellsobtained by immunizing a transgenic animal comprising a repertoire ofhuman antibody genes with an antigen such as a protein,protein-expressing cells, or a cell lysate thereof (WO92/03918,WO93/2227, WO94/02602, WO94/25585, WO96/33735, and WO96/34096).

Other than producing antibodies by using hybridoma, antibody-producingimmunocytes, such as sensitized lymphocytes that are immortalized byoncogenes, may also be used.

Such monoclonal antibodies can also be obtained as recombinantantibodies produced by using the genetic engineering technique (forexample, Borrebaeck, C. A. K. and Larrick, J. W., “TherapeuticMonoclonal Antibodies”, Published in the United Kingdom by MacMillanPublishers Ltd., 1990). Recombinant antibodies are produced by cloningthe encoding DNA from immunocytes, such as hybridoma orantibody-producing sensitized lymphocytes, incorporating this into asuitable vector, and introducing this vector into a host to produce theantibody. The present invention encompasses such recombinant antibodiesas well.

Moreover, the antibody of the present invention may be an antibodyfragment or a modified-antibody, so long as it binds to a protein of theinvention. For example, Fab, F(ab′)₂, Fv, or single chain Fv in whichthe H chain Fv and the L chain Fv are suitably linked by a linker (scFv,Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883, 1988) can begiven as antibody fragments. Specifically, antibody fragments areproduced by treating antibodies with enzymes, for example, papain,pepsin, and such, or by constructing a gene encoding an antibodyfragment, introducing this into an expression vector, and expressingthis vector in suitable host cells (for example, Co et al., J. Immunol.,152:2968-2976, 1994; Better et al., Methods Enzymol., 178:476-496, 1989;Pluckthun et al., Methods Enzymol., 178:497-515, 1989; Lamoyi, MethodsEnzymol., 121:652-663, 1986; Rousseaux et al., Methods Enzymol.,121:663-669, 1986; Bird et al., Trends Biotechnol., 9:132-137, 1991).

As modified antibodies, antibodies bound to various molecules such aspolyethylene glycol (PEG) can be used. The antibody of the presentinvention encompasses such modified antibodies as well. To obtain such amodified antibody, chemical modifications are done to the obtainedantibody. These methods are already established in the field.

The antibody of the invention may be obtained as a chimeric antibody,comprising non-human antibody-derived variable region and humanantibody-derived constant region, or as a humanized antibody comprisingnon-human antibody-derived complementarity determining region (CDR),human antibody-derived framework region (FR), and human antibody-derivedconstant region by using conventional methods.

Antibodies thus obtained can be purified to uniformity. The separationand purification methods used in the present invention for separatingand purifying the antibody may be any method usually used for proteins.For instance, column chromatography, such as affinity chromatography,filter, ultrafiltration, salt precipitation, dialysis,SDS-polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis, and so on, may be appropriately selected and combinedto isolate and purify the antibodies (Antibodies: a laboratory manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but isnot limited thereto. Antibody concentration of the above mentionedantibody can be assayed by measuring the absorbance, or by theenzyme-linked immunosorbent assay (ELISA), etc.

Protein A or Protein G column can be used for the affinitychromatography. Protein A column may be, for example, Hyper D, POROS,Sepharose F.F. (Pharmacia), and so on.

Other chromatography may also be used, such as ion exchangechromatography, hydrophobic chromatography, gel filtration, reversephase chromatography, and adsorption chromatography (Strategies forProtein Purification and Characterization: A laboratory Course Manual.Ed. by Marshak D. R. et al., Cold Spring Harbor Laboratory Press, 1996).These may be performed on liquid chromatography such as HPLC or FPLC.

Examples of methods that assay the antigen-binding activity of theantibodies of the invention include, for example, measurement ofabsorbance, enzyme-linked immunosorbent assay (ELISA), enzymeimmunoassay (EIA), radio immunoassay (RIA), or fluorescent antibodymethod. For example, when using ELISA, a protein of the invention isadded to a plate coated with the antibodies of the invention, and next,the objective antibody sample, for example, culture supernatants ofantibody-producing cells, or purified antibodies are added. Then,secondary antibody recognizing the antibody, which is labeled byalkaline phosphatase and such enzymes, is added, the plate is incubatedand washed, and the absorbance is measured to evaluate theantigen-binding activity after adding an enzyme substrate such asp-nitrophenyl phosphate. As the protein, a protein fragment, forexample, a fragment comprising a C-terminus, or a fragment comprising anN-terminus may be used. To evaluate the activity of the antibody of theinvention, BIAcore (Pharmacia) may be used.

By using these methods, the antibody of the invention and a samplepresumed to contain a protein of the invention are contacted, and theprotein of the invention is detected or assayed by detecting or assayingthe immune complex of the above-mentioned antibody and protein.

A method of detecting or assaying a protein of the invention is usefulin various experiments using proteins as it can specifically detect orassay the proteins.

Another object of this invention is to provide a polynucleotide of atleast 15 nucleotides that is complimentary to the DNA encoding humanNR10 protein (SEQ ID NO:1, 3, or 16) or its complimentary strand.

Herein, the term “complimentary strand” is defined as one strand of adouble strand DNA composed of A:T and G:C base pairs to the otherstrand. Also, “complimentary” is defined as not only those completelymatching within a continuous region of at least 15 nucleotides, but alsohaving a homology of at least 70%, favorably 80% or higher, morefavorably 90% or higher, and most favorably 95% or higher within thatregion. The homology may be determined using the algorithm describedherein.

Probes or primers for detection or amplification of the DNA encoding aprotein of the invention, or a nucleotide or nucleotide derivative forthe suppression of the protein expression (such as antisenseoligonucleotide and ribozyme) are included in these polynucleotides.Such polynucleotides may be also used for preparing DNA chips.

The antisense oligonucleotide that hybridizes with a portion of thenucleotide sequence of any of SEQ ID NO:1, 3, and 16 is also included inthe antisense oligonucleotides of the present invention. This antisenseoligonucleotide is preferably one against at least 15 continuousnucleotides in any one of the nucleotide sequences of SEQ ID NO:1, 3 and16. More preferably, it is the antisense oligonucleotide against atleast 15 continuous nucleotides containing a translation start codon.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsinclude, for example, lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type; phosphorothioate;phosphoroamidate-modified products, and such.

The term “antisense oligonucleotide(s)” as used herein means, not onlythose in which the nucleotides corresponding to those constituting aspecified region of a DNA or mRNA are entirely complementary, but alsothose having a mismatch of one or more nucleotides, so long as the DNAor mRNA and the oligonucleotide can selectively and stably hybridizewith the nucleotide sequence of SEQ ID NO:1, 3 or 16.

The antisense oligonucleotide derivative of the present invention actsupon cells producing a protein of the invention by binding to the DNA ormRNA encoding the protein to inhibit its transcription or translation,and to promote the degradation of mRNA, and has an effect of suppressingthe function of the protein of the invention by suppressing theexpression of the protein.

The antisense oligonucleotide derivative of the present invention can bemade into an external preparation such as a liniment and a poultice bymixing with a suitable base material, which is inactive against thederivatives.

Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops, and freeze-dried agents by adding excipients, isotonicagents, solubilizers, stabilizers, preservatives, pain-killers, etc.These can be prepared using conventional methods.

The antisense oligonucleotide derivative is given to the patient bydirectly applying onto the ailing site, by injecting into the bloodvessel and such, so that it will reach the ailing site. Anantisense-mounting material can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L lysine, lipid,cholesterol, lipofectin, or derivatives of them.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

The antisense oligonucleotide derivative of the present invention isuseful in inhibiting the expression of the protein of the invention, andtherefore is useful in suppressing the biological activity of theprotein of the invention. Also, expression-inhibitors comprising theantisense oligonucleotide derivative of the present invention areuseful, because of their capability to suppress the biological activityof the protein of the invention.

Proteins of this invention are useful for screening compounds binding tothe protein. That is, the proteins are used in the method of screeningfor compounds that bind to the proteins of this invention, in which themethod comprises bringing proteins of this invention into contact with atest sample that is expected to contain a compound to bind to theproteins and selecting a compound with the activity to bind to theproteins of this invention.

Proteins of this invention used in the screening may be any ofrecombinant, natural or partial peptides. Also they may be a purifiedprotein, partial peptides thereof, or in the form of proteins expressedon the cell surface or membrane fractions. Samples to be tested are notlimited, but may be cell extracts, culture supernatants, fermentedproducts of microorganisms, extracts of marine organisms, plantextracts, purified or partially purified proteins, peptides, non-peptidecompounds, synthetic low molecular compounds, or natural compounds. Theprotein of the invention may be exposed to the sample as a purifiedprotein or soluble protein, in a form bound to a support, as a fusionprotein with another protein, in a form expressed on the surface of cellmembrane, or as membrane fractions.

A protein of the invention may be used to screen for proteins that bindto the protein (such as ligands) using a variety of methods known to oneskilled in the art. These screening can be carried out, for example, bythe immunoprecipitation method. Specifically, the method can be carriedout as follows. The gene encoding the protein of this invention isexpressed by inserting the gene into a vector for foreign geneexpression like pSV2neo, pcDNA I, pCD8, and such, and expressing thegene in animal cells, etc. Any generally used promoters may be employedfor the expression, including the SV40 early promoter (Rigby InWilliamson (ed.), Genetic Engineering, Vol. 3. Academic Press, London,p. 83-141, 1982), EF-1α promoter (Kim et al., Gene, 91:217-223, 1990),CAG promoter (Niwa et al., Gene, 108:193-200, 1991), RSV LTR promoter(Cullen, Methods in Enzymology, 152:684-704, 1987), SR α promoter(Takebe et al., Mol. Cell. Biol., 8:466, 1988), CMV immediate earlypromoter (Seed et al., Proc. Natl. Acad. Sci. USA, 84:3365-3369, 1987),SV40 late promoter (Gheysen et al., J. Mol. Appl. Genet., 1:385-394,1982), Adenovirus late promoter (Kaufman et al., Mol. Cell. Biol.,9:946, 1989), HSV TK promoter, etc. Transfer of a foreign gene intoanimal cells for its expression can be performed by any of the followingmethods, including the electroporation method (Chu et al., Nucl. AcidRes., 15:1311-1326, 1987), the calcium phosphate method (Chen et al.,Mol. Cell. Biol., 7:2745-2752, 1987), the DEAE dextran method (Lopata etal., Nucl. Acids Res., 12:5707-5717, 1984; Sussman et al., Mol. Cell.Biol., 4:1642-1643, 1985), the lipofectin method (Derijard, Cell.,7:1025-1037, 1994; Lamb et al., Nature Genetics, 5:22-30, 1993;Rabindran et al., Science, 259:230-234, 1993), etc.

A protein of the present invention can be expressed as a fusion proteinhaving the recognition site for a monoclonal antibody by introducing arecognition site (epitope) for a monoclonal antibody, the specificity ofwhich has been established, into the N- or C-terminal of the protein ofthis invention. For this purpose, a commercial epitope-antibody systemcan be utilized (Jikken Igaku, Experimental Medicine, 13:85-90, 1995).Vectors are commercially available which are capable of expressingfusion proteins with β-galactosidase, maltose-binding protein,glutathione S-transferase, green fluorescence protein (GFP), and such,via the multi-cloning site.

To minimize the alteration of the properties of the protein of thisinvention due to the formation into a fusion protein, a method has beenreported to prepare a fusion protein by introducing only a small epitopeportion comprising several to ten amino acid residues. For example, theepitopes of polyhistidine (His-tag), influenza hemagglutinin (HA), humanc-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV-tag),E-tag (epitope on the monoclonal phage), and such, and monoclonalantibodies to recognize these epitopes can be utilized as theepitope-antibody system for screening proteins binding to the protein ofthis invention (Jikken Igaku, Experimental Medicine, 13:85-90, 1995).

In the immunoprecipitation, immune complexes are formed by adding theseantibodies to the cell lysate prepared using suitable surfactants. Thisimmune complex consists of a protein of this invention, a proteincapable of binding to the protein, and an antibody. Theimmunoprecipitation can also be performed using an antibody to a proteinof this invention besides antibodies to the above-described epitopes. Anantibody to a protein of this invention can be prepared by inserting agene encoding a protein of this invention into an appropriate expressionvector for E. coli to express it in the bacterium, purifying the proteinthus expressed, and immunizing rabbits, mice, rats, goats, chicken, andsuch, with the purified protein. The antibody can also be prepared byimmunizing the above-described animals with partial peptides of theprotein of this invention.

Immune complexes can be precipitated using, for example, Protein ASepharose and Protein G Sepharose in case where the antibody is a murineIgG antibody. In addition, in the case where the protein of thisinvention is prepared as a fusion protein with the epitope of, forexample, GST, and such, the immune complex can be formed using asubstance that specifically binds to these epitopes, such asglutathione-Sepharose 4B, and such, giving the same result as in thecase where the antibody for the protein of this invention is used.

Immune precipitation, in general, may be carried out according to, orfollowing the method described in the literature (Harlow, E. and Lane,D.: Antibodies, pp. 511-552, Cold Spring Harbor Laboratory publications,New York, 1988).

SDS-PAGE is generally used for the analysis of immunoprecipitatedproteins, and bound proteins can be analyzed based on the molecularweights of proteins using a gel of an appropriate concentration. In thiscase, although proteins bound to a protein of this invention, ingeneral, are hardly detectable by the usual protein staining method,such as Coomassie staining and silver staining, the detectionsensitivity can be improved by culturing cells in a medium containingthe radio isotope-labeled ³⁵S-methionine and ³⁵S-cysteine to labelproteins inside the cells, and detecting the labeled proteins. Once themolecular weight of the protein is determined, the desired protein canbe purified directly from SDS-polyacrylamide gel and sequenced.

In addition, screening of proteins binding to a protein of the presentinvention can be also performed using the West-western blotting method(Skolnik et al., Cell, 65:83-90, 1991). Specifically, cDNA is isolatedfrom cells, tissues and organs (for example, tissue, cell or cultivatedcell of heart, placenta, testis, thymus, peripheral leukocyte, etc.) inwhich protein binding to the protein of this invention is expected to beexpressed, and transferred into a phage vector (for example, λgt11,ZAPII etc.), to prepare a cDNA library, which is then expressed onplates coated with a growth medium. The protein thus expressed is fixedon a filter, which is then reacted with the labeled, purified protein ofthis invention, and plaques expressing proteins bound to the protein ofthis invention can be detected by the label. Methods for labeling aprotein of this invention include methods utilizing the binding activityof biotin and avidin, methods utilizing antibodies specifically bindingto the protein of this invention, or peptides or polypeptides (forexample, GST etc.) fused with the protein of this invention, methodsutilizing the radioisotopes, methods utilizing fluorescence, etc.

Further, another embodiment of the screening method of this invention isexemplified by a method utilizing the 2-hybrid system using cells(Fields et al., Trends. Genet., 10:286-292, 1994; Dalton et al., Cell,68:597-612, 1992; “MATCHMARKER Two-Hybrid System”, “MammalianMATCHMARKER Two-Hybrid Assay Kit”, “MATCHMARKER One-Hybrid System”(Clonetech); “HybriZAP Two-Hybrid Vector system” (Stratagene)). In thetwo-hybrid system, a protein of the invention may be fused to the DNAbinding domain of SRF or GAL4, and expressed in yeast. A cDNA library isconstructed from cells predicted to express proteins that bind to theprotein of the present invention, wherein the cDNA library isconstructed in such a way that the proteins are expressed as fusionproteins with transcription activation regions of VP 16 or. The cDNAlibrary is transfected into the above yeast, and then positive clonesare be detected to isolate the cDNA derived from the library (Expressionof a protein that binds to the protein of the invention in yeast leadsto the binding of the two proteins, and results in the activation of thereporter gene, which allows to detect positive clones). The proteinencoded by the isolated cDNA may be obtained by introducing the cDNAinto E. coli and expressing it therein. Thus, it is possible to prepareproteins that binds to a protein of the invention and genes encodingthem. The reporter gene used in the two-hybrid system may be such asHIS3, Ade2, LacZ, CAT, luciferase, or PAI-I (plasminogen activatorinhibitor type I), but is not limited thereto.

Screening for compounds, which bind to a protein of this invention, canbe also carried out using affinity chromatography. For example, theprotein of this invention is immobilized on a carrier in the affinitychromatography column, to which a test sample, which is expected toexpress a protein binding to the protein of this invention, is applied.Samples may be cell extracts, cell lysates, or else. After applying thetest sample, the column is washed, and protein which binds to theprotein of the invention can be obtained.

The obtained protein may be analyzed for its amino acid sequence tosynthesize oligonucleotide probes, which may be used to screen a cDNAlibrary to obtain a DNA encoding the protein.

A biosensor that utilizes surface plasmon resonance may be used todetect or measure the bound compound. Such sensor (as BIAcore(Pharmacia)) may enable to observe the interaction at real time using asmall amount of protein without the need of labeling. Thus, it ispossible to assess the interaction between the protein of the inventionand samples using such biosensor as BIAcore.

Moreover, compounds that bind to a protein of the invention (includingagonists and antagonists), which compounds are not always proteins, maybe isolated using a variety of methods known to one skilled in the art.For instance, the protein of the invention may be fixed and exposed tosynthetic compounds, a bank of natural compounds, or a random phagepeptide library to screen a molecule that binds to the protein.Alternatively, high throughput screening using combinatorial chemistrymay be performed (Wrighton et al., Science, 273:458-464, 1996; Verdine,Nature, 384:11-13, 1996); Hogan Jr., Nature, 384:17-9, 1996).

Screening of a ligand that binds to a protein of the invention may beperformed as follows. The extracellular domain of the protein of theinvention is fused to the intracellular domain including thetransmembrane domain of a hemopoietin receptor protein that has a knownsignal transducing ability to prepare a chimeric receptor. The chimericreceptor may be expressed on the cell surface of an appropriate cellline, favorably a cell line that is capable of growing only in thepresence of an appropriate growth factor (growth factor-dependent cellline). Then, the cell line may be cultured in medium supplemented with asample material in which a variety of growth factors, cytokines, orhematopoietic factors might be expressed. According to this method, thegrowth factor-dependent cell line can only survive and proliferate whenthe sample contains an appropriate ligand that specifically binds to theextracellular domain of the protein of the invention. The knownhemopoietin receptor, such as thrombopoietin receptor, erythropoietinreceptor, G-CSF receptor, and gp130 may be used. The partner forconstructing a chimeric receptor for the screening system of the presentinvention is not limited to the above receptors as long as itsintracellular domain provides a structure necessary for the signaltransduction activity. The growth factor-dependent cell line may be anIL-3-dependent cell line such as BaF3 or FDC-P1.

In a rare case, the ligand that specifically binds to a protein of theinvention may not be a soluble protein but a membrane-bound protein. Inthis case, screening can be performed using a protein comprising onlythe extracellular domain of the protein of the invention, or a fusionprotein in which the extracellular domain is attached to a part of othersoluble proteins. Such proteins are labeled before they are used formeasuring the binding with the cells that are expected to express theligand. The former protein comprising only the extracellular domain maybe a soluble receptor protein artificially constructed throughintroducing a stop codon into the N-terminal region of the transmembranedomain, or a soluble protein such as NR10.2. The latter fusion proteinmay be a protein in which the Fc region of immunoglobulin G, or FLAGpeptide is attached to the C-terminus of the extracellular domain. Theselabeled soluble proteins may be also useful for detection by thewest-western method.

A chimeric protein of the extracellular domain of a protein of theinvention and the Fc region of an antibody (such as human IgG) may bepurified using a Protein A column. Such antibody-like chimeric proteinretains the ligand binding ability. Thus, the protein may beappropriately labeled with an isotope and so on, and used for thescreening of a ligand (Suda et al., Cell, 175:1169-1178, 1993). Somecytokines such as molecules of the TNF family primarily exist in amembrane bound form, so such ligands may be isolated by exposing theantibody-like chimeric protein to a variety of cells and selecting cellsby the binding ability to the protein. Alternatively, ligands may beisolated according to the same method by using cells to which a cDNAlibrary is introduced. Furthermore, the antibody-like chimeric proteinmay be also used as an antagonist.

The compounds obtained by the above screening may be a candidate fordrugs that activate or inhibit the activity of a protein of theinvention. It is possible to use such compounds for the treatment ofdiseases arising from abnormal expression or functional disorder of aprotein of the present invention. The compound obtained by using thescreening method of the invention includes compounds resulting from themodification of the compound having the activity to bind to the proteinof the invention by adding, deleting, and/or replacing a part of thestructure.

When using the isolated compound or a protein of the present invention(decoy type (soluble form)) as a pharmaceutical for humans and othermammals, for example, mice, rats, guinea-pigs, rabbits, chicken, cats,dogs, sheep, pigs, cattle, monkeys, sacred baboons, chimpanzees, theisolated compound can be directly administered or can be formulated intoa dosage form using known pharmaceutical preparation methods. Forexample, according to the need, the drugs can be taken orally, assugar-coated tablets, capsules, elixirs and microcapsules, orparenterally, in the form of injections of sterile solutions,suspensions with water, or any other pharmaceutically acceptable liquid.For example, the compounds can be mixed with pharmacologicallyacceptable carriers or medium, specifically, sterilized water,physiological saline, plant-oil, emulsifiers, solvents, surfactants,stabilizers, flavoring agents, excipients, vehicles, preservatives andbinders, in a unit dose form required for generally accepted drugimplementation. The amount of active ingredients in these preparationsmakes a suitable dosage acquirable within the indicated range.

Examples for additives which can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and gum arabic;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin and alginic acid; lubricants such as magnesium stearate;sweeteners such as sucrose, lactose or saccharin; flavoring agents suchas peppermint, Gaultheria adenothrix oil and cherry. When the unitdosage form is a capsule, a liquid carrier, such as oil, can also beincluded in the above ingredients. Sterile composites for injections canbe formulated following normal drug implementations using vehicles suchas distilled water used for injections.

For example, physiological saline, glucose, and other isotonic liquidsincluding adjuvants, such as D-sorbitol, D-mannnose, D-mannitol, andsodium chloride, can be used as aqueous solutions for injections. Thesecan be used in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as Polysorbate 80™ andHCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol assolubilizers; may be formulated with a buffer such as phosphate bufferand sodium acetate buffer; a pain-killer such as procaine hydrochloride;a stabilizer such as benzyl alcohol, phenol; and an anti-oxidant. Theprepared injection is generally filled into a suitable ampule.

Methods well known to one skilled in the art may be used to administerthe pharmaceutical compound to patients, for example as intraarterial,intravenous, percutaneous injections and also as intranasal,transbronchial, intramuscular or oral administrations. The dosage andmethod for administration vary according to the body-weight and age ofthe patient, the administration method, and such, but one skilled in theart can suitably select them. If said compound is encodable by a DNA,said DNA can be inserted into a vector for gene therapy to perform thetherapy. The dosage and method for administration vary according to thebody-weight, age, symptoms of a patient, and so on, but one skilled inthe art can select them suitably.

For example, the dose of the protein (decoy type (soluble form)) mayvary depending on the patient, target organ, disease type, and methodfor administration. However, it may be injected to a normal adult (bodyweight, 60 kg) at a dose of 100 μg to 10-20 mg per day.

For example, although there are some differences according to thesymptoms, the dose of a compound that binds with the protein of thepresent invention, or a compound that inhibits the activity of theprotein of this invention is about 0.1 mg to about 100 mg per day,preferably about 1.0 mg to about 50 mg per day, and more preferablyabout 1.0 mg to about 20 mg per day, when administered orally to astandard adult (weight 60 kg).

When the protein is administered parenterally in the form of aninjection to a standard adult (weight 60 kg), although there are somedifferences according to the patient, target organ, symptoms and methodof administration, it is convenient to intravenously inject a dose ofabout 0.01 mg to about 30 mg per day, preferably about 0.1 to about 20mg per day, and more preferably about 0.1 to about 10 mg per day. Also,in the case of other animals, it is possible to administer an amountconverted to 60 kg of body-weight or surface area.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications and patents cited herein are incorporated by referencein their entirety.

DESCRIPTION OF DRAWINGS

FIG. 1 is a representation of the nucleotide sequence of AQ022781 (SEQID NO:34) identified in the gss database. The deduced amino acidsequence (SEQ ID NO:35) is shown under the predicted exon sequence. TheYR motif and WS motif that were used as the target are boxed. Two “n” inthe nucleotide sequence are also boxed.

FIG. 2 is a representation of partial amino acid sequences of NR10(amino acid residues 198-238, 201-237, 196-237, 189-238, and 196-239 ofSEQ ID NO:4, respectively) found in the sequence of AQ022781 (SEQ IDNO:35, which is part of SEQ ID NO:4), and those of known hemopoietinreceptors having homology thereto. Identical residues are boxed withshadow, and similar residues are shadowed. Gap spaces are underlined.Known hemopoietin receptors are, from top, human gp130 (GenBankAccession No. NM002184.1; IL6ST; SEQ ID NO:36), human LIF receptor(GenBank Accession No. NM002310.1; LIFR; SEQ ID NO:37), human OncostatinM receptor β subunit (GenBank Accession No. NM003999.1; OSMR; SEQ IDNO:38), human IL-12 receptor β2 subunit (GenBank Accession No.NM001559.1; IL12RB2; SEQ ID NO:39), and human NR6 (GenBank Accession No.AC003112; SEQ ID NO:40).

FIG. 3 is a representation of the nucleotide sequence of the full lengthNR10.1 cDNA (SEQ ID NO:1) that was obtained by combining the 5′- and3′-RACE products. The deduced amino acid sequence encoded by NR10.1 isalso shown (SEQ ID NO:2). The amino acid sequence predicted to be thesecretion signal sequence is underlined. The predicted transmembranedomain is shadowed. Conserved cysteine residues and the WS motif areboxed.

FIG. 4 is a continuation of FIG. 3.

FIG. 5 is a continuation of FIG. 4.

FIG. 6 shows the nucleotide sequence of the full length NR10.2 cDNA (SEQID NO:3) that was obtained by combining the 5′- and 3′-RACE products.The deduced amino acid sequence encoded by NR10.2 is also shown (SEQ IDNO:4). The predicted secretion signal sequence is underlined. Conservedcysteine residues and the WS motif are boxed.

FIG. 7 is a continuation of FIG. 6.

FIG. 8 shows photographs demonstrating the result of RT-PCR analysis ofthe expression pattern of the NR10.1 gene in human organs.

FIG. 9 shows photographs demonstrating the result of RT-PCR analysis ofthe expression pattern of the NR10.2 gene in human organs.

FIG. 10 shows a photograph demonstrating the result of quantification ofthe NR10.1 gene expression in human organs by Southern blotting.

FIG. 11 shows a photograph demonstrating the result of quantification ofthe NR10.2 gene expression in human organs by Southern blotting.

FIG. 12 is a schematic illustration of the structure of the protein tobe expressed from the expression vector construct.

FIG. 13 shows the nucleotide sequence of the full length NR10.3 cDNA(SEQ ID NO:16). The deduced amino acid sequence encoded by NR10.3 isalso shown (SEQ ID NO:17). The predicted secretion signal sequence isunderlined. The amino acid sequence predicted to be the transmembranedomain is colored. Conserved cysteine residues and the WS motif areboxed.

FIG. 14 is a continuation of FIG. 13.

DETAILED DESCRIPTION

This invention will be explained in detail below with reference toexamples, but it is not construed as being limited thereto.

Example 1 Isolation of NR10.1 and NR10.2 Genes (1) BLAST Search

The inventors aimed at finding another motif conserved among thehemopoietin receptor family, in addition to the Trp-Ser-Xaa-Trp-Ser (SEQID NO:22) motif (WS motif), in order to design an oligonucleotide probeincluding both motifs together. The inventors examined the sequence ofother regions for another motif. As a result, they found a tyrosine orhistidine residue in the extracellular domain of the family proteins,located 13 to 27 amino acids upstream of the WS motif, that is conservedat high frequency. They further examined the six amino acid residueslocated to the C-terminus from the Tyr/His residue for a consensussequence that appears with a high-frequency, and found the amino acidfollowing sequence:(Tyr/His)-Xaa-(Hydrophobic/Ala)-(Gln/Arg)-Hydrophobic-Arg (referred toas the YR motif in the following). However, the YR motif is notconsidered as a perfect consensus sequence, and also the combination ofnucleotide sequences that can encode the motif is really complicated.Thus, it seemed very difficult to synthesize all the nucleotidesequences that encode the amino acid sequence and provide them as theprobe for hybridization, a practical method of screening, or as theprimer for RT-PCR.

Accordingly, the inventors examined for a specific method of screeningfor a novel hemopoietin receptor using the above motifs as the probe. Asa result, they found it reasonable to perform a database search oncomputer using a query composed of a partial amino acid sequence ofknown hemopoietin receptors, a fragment including both motifs.

First, amino acid sequences that fulfilled the necessary condition tocontain both motifs were designed to prepare a query for databasesearch. Although the receptor family normally contains a spacer of 7 to10 amino acids between the motifs, the spacer was fixed to 10 aminoacids by taking average. It was expected that even if the length ofspacer in target genes were different from that in the query, the gapwould be filled by space so that it would not interfere the search.Moreover, the number of undetermined residues was minimized so as toincrease the quality of the sequence and improve the sensitivity ofdetection. Thus, based on the sequence that appeared frequently in knownhemopoietin receptors, three patterns were designed tentatively for theYR motif, two residues on both ends of the spacer, and the residues atthe center and the C-terminus of the WS motif, respectively, as in Table1.

TABLE 1 YR motif spacer amino acids WS motif YTVQVR AR XXXXXX GT WSEWSP(SEQ ID NO: 23) (SEQ ID NO: 26) (SEQ ID NO: 29) YEARVR VQ XXXXXX GYWSDWSE (SEQ ID NO: 24) (SEQ ID NO: 27) (SEQ ID NO: 30) YSLQLR CK XXXXXXGI WSPWSQ (SEQ ID NO: 25) (SEQ ID NO: 28) (SEQ ID NO: 31)

Combining the YR motif, spacer and the WS motif described in Table 1gives 27 different queries. The queries were used to search the nrdatabase in GenBank using the TblastN program (Advanced TblastN 2.0.8).Parameters for search were set as Expect value=100, Descriptions=100,and Alignments=100. As a result, many of known hemopoietin receptorswere identified positive, confirming that the method was workingcorrectly. Then, the same queries were used to search on the ESTdatabase as well as the gss and htgs database in order to detect asequence that could encode a novel hemopoietin receptor. However, theresult did not yield any positive clones that appeared novel. It wasconsidered that the limited variety of the above-mentioned 27 queries isthe cause of the result. Accordingly, further preparation of a varietyof sequences for the query was attempted, but the combination of thesequence became too complicated to continue the preparation manually.After all, the inventors decided to use partial amino acid sequences ofknown hemopoietin receptors that were fragmented so as to include bothof the YR and WS motifs in order to prepare a query for database search.

Comparison of the genomic structure of the receptor family revealed thatthe YR and WS motifs are contained within a single exon in all examinedknown hemopoietin receptors. This suggests that the continuity andcompatibility of both motifs may be also retained in the genomicsequence. Therefore, it was expected that the exon of a knownhemopoietin receptor encoding both motifs are effective as the query tosearch for the target gene on the EST database, and the genomic databaseas well. Herein, human gp130 and human LIF receptor sequences were usedas the known hematopoietin receptor sequence, because their structureshave a relatively high similarity among the receptor family, and thesimilarity is expected to be shared in the target novel receptor. Whilethe sequences of human gp130 and human LIF receptor were already known,the inventors used the amino acid sequence encoded by the cDNA that hadbeen isolated by the inventors themselves using plaque hybridization andRT-PCR with a probe encoding the WS motif.

Based on the genomic structure, it is known that hemopoietin receptorsare to contain an exon encoding the YR and WS motifs having a length of50 to 70 amino acids. Accordingly, 29 amino acids to the N-terminus and30 amino acids to the C-terminus from the first Tyr residue in the YRmotif, a total of 60 amino acids were cut out of the sequence of humangp130 and human LIF receptor to prepare a query sequence forconvenience' sake. The LIF receptor contains two WS motifs, and thesecond (on the C-terminal side) WS motif was selected taking intoaccount the conservation of the YR motif. The above queries were used tosearch on the gss (Genomic Survey Sequence) and htgs database in GenBankusing TblastN (Advanced TblastN 2.0.8). Parameters were set as Expectvalue=50, Descriptions=100, and Alignments=100.

The length of the selected query sequence, 60 amino acids, was notexactly the same as that of the actual exon sequence. However, takinginto account that the length of this exon in known hemopoietin receptorgenes differ somewhat according to each gene, and by taking theconservation of both YR and WS motifs as the index into muchconsideration it was decided that the difference may not interfere withthe search. The gss and htgs database was used because these genomicsequences has not been fully analyzed due to its complexity, and thus,it was expected that they are suitable for identifying novel receptorgenes. Since the queries were longer than the previous 27 artificialqueries, parameters “Expect value=50, Descriptions=100, andAlignments=100” were set to reduce the sensitivity of detection so as toavoid increase of false positive clones that have homology to a regionother than the motifs. Thus, it was expected that this enables detectionof target genes by suppressing detection of such false positive clonesshowing homology at sequences other than the target motif sequence.

As a result, the search resulted in many hits of false positive clones,and those clones in which both YR and WS motifs were not encoded in thesame reading frame, or that contained a stop codon between the motifswere discarded. Also, those clones containing only the YR motif but notthe WS motif were discarded, because, as mentioned above, the YR motifis not a completely established consensus sequence. Therefore, theconservation of the WS motif was considered predominant. As a result, asingle clone containing the human genomic sequence (GenBank AccessionNo. AQ022781) expected to encode a part of a novel hemopoietin receptorgene was selected, and the gene was named NR10.

AQ022781 (SEQ ID NO:34) is the terminal sequence of a BAC cloneconsisting of 459 bp, deposited in the gss database. It was the onlyclone that was also positive in both searches using partial amino acidsequences of human gp130 or LIF receptor as the query respectively. Itwas presumed that the reliability of the sequence might be low due theexistence of two “n” in the middle and the nature of the depositionsystem of the Genomic

Survey Sequence. Nevertheless, as shown in FIG. 1, a splice consensussequence could be recognized as the “ag” sequence following the “c/t”rich sequence at 175th to the 218th bases, and it was predictable thatit contains an exon starting from “atg” following the splice consensussequence. Then, the predicted exon sequence was used to search on the nrdatabase in GenBank using BlastX (Advanced BlastX 2.0.8). The resultsrevealed that the exon has homology to many known hemopoietin receptorgenes as shown in FIG. 2. The result was: (1) AQ022781 (SEQ ID NO:35)contains a YR motif, sequence YVIALR (SEQ ID NO:32), and that itretained a complete WS motif, sequence WSDWS (SEQ ID NO:33); (2) showinghomology with several known hemopoietin receptors, and (3) both of thetwo Ser residues in the WS motif are encoded by AG(C/T). And thus, itwas predicted that the gene could encode a novel hemopoietin receptorgene. The codon for Ser in the WS motif is generally AG(C/T) in most ofthe known hemopoietin receptors, but the second Ser residue in the EPOreceptor, TPO receptor, and mouse IL-6 receptor is encoded by TCN.Indeed, most of the false positive clones containing by chance a WSmotif-like sequence, the second Ser was mostly encoded by TCN. Thus, theSer residue encoded by the AG(C/T) codon could be used as a marker forselection of positive clones. Accordingly, specific oligonucleotideprimers were designed from the predicted exon sequence on AQ022781, andused for 5′-RACE and 3′-RACE method as below.

(2) Design of Oligonucleotide Primers

As described in (1), exon sites were predicted on AQ022781 sequences,and these sequences were used to design the following oligonucleotideprimers specific for NR10. Three sense primers (NR10-S1, NR10-S2, andNR10-S3; oriented downstream) and three antisense primers (NR10-A1,NR10-A2, and NR10-A3; oriented upstream) were synthesized using the ABI394 DNA/RNA synthesizer under a condition to attach a trityl group tothe 5′-terminus. Then, the products were purified using an OPC column(ABI #400771) to obtain full-length primers.

NR10-S1: (SEQ ID NO: 5) 5′-ATG GAA GTC AAC TTC GCT AAG AAC CGT AAG-3′NR10-S2: (SEQ ID NO: 6) 5′-CCA AAC GTA CAA CCT CAC GGG GCT GCA ACC-3′NR10-S3: (SEQ ID NO: 7) 5′-GTC ATA GCT CTG CGA TGT GCG GTC AAG GAG-3′NR10-A1: (SEQ ID NO: 8) 5′-agt agc ttg cgT TCT TCC TCA GCT ATT CCC-3′NR10-A2: (SEQ ID NO: 9) 5′-CTT TGA CTC CTT GAC CGC ACA TCG CAG AGC-3′NR10-A3: (SEQ ID NO: 10) 5′-GGT TGC AGC CCC GTG AGG TTG TAC GTT TGG-3′

The “n” at position 376 in AQ022781 sequence (FIG. 1) was assigned to bebase “c” to design the primer sequences above, and thus, correspondingbase at position 11 in NR10-A1 primer sequence was designed “g”.According to the analysis of the consensus sequence for splicing theminimal exon on AQ022781 sequence was predicted to be starting from base“a” at position 211 to base “c” at position 399, the intron startingfrom the next “gt” sequence. However, the analysis of 3′-RACE productsas described later revealed that the intron starts from the base “n” atposition 376 or from base “g” at position 377. Therefore, as a result,the 11 bases shown in small caps of NR10-A1 primer sequence above can'tbind correctly during PCR, while the corresponding sequence is nottranscribed into mRNA. However, PCR reactions proceeded correctly,probably because the other 19 bases, the 3′-terminal sequences, werecapable of annealing specifically.

(3) Cloning of the C-Terminus cDNA by 3′-RACE Method

In order to isolate the full-length cDNA of NR10, 3′-RACE PCR wasperformed using NR10-S1 and NR10-S2 primers described in (2) for primaryand secondary PCR, respectively. PCR experiment was performed usingHuman Fetal Liver Marathon-Ready cDNA Library (Clontech #7403-1) as thetemplate, and Advantage cDNA Polymerase Mix (Clontech #8417-1) on athermal cycler (Perkin Elmer Gene Amp PCR System 2400). Under thefollowing conditions, as a result, PCR products showing two differentsizes by alternative splicing were obtained.

Condition of the primary PCR was as follows: a single cycle of “94° C.for 4 min”, 5 cycles of “94° C. for 20 sec, and 72° C. for 100 sec”, 5cycles of “94° C. for 20 sec, and 70° C. for 100 sec”, 28 cycles of “94°C. for 20 sec, and 68° C. for 100 sec”, a single cycle of 72° C. for 3min, and termination at 4° C.

Condition of the secondary PCR was as follows: a single cycle of “94° C.for 4 min”, 5 cycles of “94° C. for 20 sec, and 70° C. for 100 sec”, 25cycles of “94° C. for 20 sec, and 68° C. for 100 sec”, a single cycle of72° C. for 3 min, and termination at 4° C.

Two amplification products were obtained by the PCR and both of themwere subcloned into the pGEM-T Easy vector (Promega #A1360), and thenucleotide sequences were determined. The transformation of the PCRproduct into the pGEM-T Easy vector was performed using T4 DNA ligase(Promega #A1360) in a reaction of 12 hrs at 4° C. Recombinants of thePCR products and pGEM-T vector were obtained by the transformation of E.coli DH5a strain (TOYOBO #DNA-903). Recombinants were selected by usingInsert Check Ready Blue (TOYOBO #PIK-201). The nucleotide sequences weredetermined using the BigDye Terminator Cycle Sequencing SF ReadyReaction Kit (ABI/Perkin Elmer #4303150) and by analyzing with the ABIPRISM 377 DNA Sequencer. Nucleotide sequences of the whole insertfragment of six independent clones were determined. As a result, theywere divided into two groups, each composed of 3 clones, based on thedifference in length and sequence of the base pairs. It was confirmedthat the difference of the product resulted from alternative splicing,and both of the obtained sequences are partial nucleotide sequences ofNR10. The cDNA clone possibly encoding the long ORF including thetransmembrane region was named as NR10.1, and the other possiblyencoding a short ORF without the transmembrane region was named asNR10.2.

(4) Cloning of the N-Terminal cDNA by 5′-Race

In order to isolate the full-length cDNA of NR10, 5′-RACE PCR wasperformed using NR10-A1 and NR10-A2 primers of Example 2 for primary andsecondary PCR, respectively. As in 3′-RACE, PCR experiment was performedusing Human Fetal Liver Marathon-Ready cDNA Library as the template, andAdvantage cDNA Polymerase Mix on a thermal cycler (Perkin Elmer Gene AmpPCR System 2400). Under the same condition to those described in (3),PCR products of three different sizes were obtained. All of the threeproducts were subcloned into the pGEM-T Easy vector as described aboveto determine the nucleotide sequence. The transformation of the PCRproducts into the pGEM-T Easy vector was performed using T4 DNA ligasein a reaction for 12 hrs at 4° C. The recombinants of the PCR productsand pGEM-T vector were obtained by transformation of E. coli DH5astrain, and selection of the recombinants were done using Insert CheckReady Blue as described above. The nucleotide sequences were alsodetermined as above using the BigDye Terminator Cycle Sequencing SFReady Reaction Kit and the ABI PRISM 377 DNA Sequencer for analysis. Theresult revealed that the obtained three 5′-RACE products with differentsizes were derived from the same mRNA transcript. The difference in sizewas due to incomplete extension reaction in the 5′-RACE and thepossibility was denied that they were derivatives of alternativesplicing. Yet, even the cDNA clone with the longest extension productamong the three 5′-RACE products did not contain the 5′-terminus of thefull-length sequence. Furthermore, another attempt using NR10-A2 andNR10-A3 primers of (2) for primary and secondary PCR, respectively,ended in a similar result. Accordingly, in order to perform another5′-RACE elongation reaction, new oligonucleotide primers were designedproximally to the N-terminus of the obtained nucleotide sequence. Twoantisense primers, NR10-A4 and NR10-A5, (upstream orientation) as belowwere prepared according to Example 2.

(SEQ ID NO: 11) NR10-A4: 5′-ATC AGA TGA AAC AGG CGC CAA CTC AGG-3′ (SEQID NO: 12) NR10-A5: 5′-TGG TTT CAC ACG GAA AAT CTT AGG TGG-3′

As described above, 5′-RACE PCR was performed using Human Fetal LiverMarathon-Ready cDNA Library as the template, and NR10-A4 and NR10-A5primer for primary and secondary PCR, respectively. Conditions for PCR,method of subcloning, and method for determining the nucleotide sequencewere as those described in (3). However, results of the sequencedetermination revealed that again only incomplete elongation products,in which the extension reaction stopped at the same site as by the5′-RACE PCR using NR10-A1, NR10-A2, and NR10-A3 primers above, wereobtained. It was possible that NR10 mRNA forms a tertiary conformationat that position so that it blocks the synthesis of primary cDNA strand.There is also the possibility that the nucleotide sequence of theupstream region from that position might have a high G/C content, whichcould block the PCR reaction. Anyway, it might be the case that thequality of the library used to prepare the cDNA library might have beenlow. Accordingly, the template for PCR was substituted with HumanPlacenta Marathon-Ready cDNA library (Clontech #7411-1) as described inthe following. This human Placenta derived material was chosen accordingto the result tissue distribution of NR10 gene by RT-PCR analysisdescribed later.

(5) Cloning of the N-Terminal cDNA Through Continuous Extension by5′-Race

To isolate the N-terminal sequence of a cDNA clone corresponding to thefull length NR10, 5′-RACE PCR was performed using NR10-A4 and NR10-A5primers of (4) for primary and secondary PCR, respectively. HumanPlacenta Marathon-Ready cDNA library was used as the template due toreasons mentioned above. Advantage cDNA Polymerase Mix was used in thePCR experiment. 5′-RACE PCR was conducted using the thermal cyclerPerkin Elmer Gene Amp PCR System 2400 under the following conditions toobtain a PCR product of single size.

The condition for primary PCR was as follows: a single cycle of “94° C.for 4 min”, 5 cycles of “94° C. for 20 sec, and 72° C. for 2 min”, 5cycles of “94° C. for 20 sec, and 70° C. for 2 min”, 28 cycles of “94°C. for 20 sec, and 68° C. for 90 sec”, a single cycle of 72° C. for 3min, and termination at 4° C.

The condition for secondary PCR was as follows: a single cycle of “94°C. for 4 min”, 5 cycles of “94° C. for 20 sec, and 70° C. for 90 sec”,25 cycles of “94° C. for 20 sec, and 68° C. for 90 sec”, a single cycleof 72° C. for 3 min, and termination at 4° C.

The obtained PCR product was subcloned into pGEM-T Easy vector asdescribed in Example 3, and the nucleotide sequence was determined. Thenucleotide sequences of the whole insert fragment from 4 independentclones of transformants revealed that the clones contain the N-terminalsequence of the full length NR10 cDNA clone. Then, the nucleotidesequence determined by the 5′RACE-PCR and those determined by 3′-RACE in(3) were combined to finally obtain the full length nucleotide sequenceof full length NR10.1 and NR10.2 cDNA. The nucleotide sequencedetermined for NR10.1 cDNA (SEQ ID NO:1) and the amino acid sequenceencoded by the sequence (SEQ ID NO:2) are shown in FIGS. 3 to 5. Thenucleotide sequence determined for NR10.2 cDNA (SEQ ID NO:3) and theamino acid sequence encoded by the sequence (SEQ ID NO:4) are shown inFIGS. 6 and 7.

According to the determination of the full-length nucleotide sequence ofNR10 cDNA, it was revealed that the “n” at position 281 of AQ022781(FIG. 1) was actually “t”. Whereas, the “n” at position 376 was notdetermined because the intron starts from the base around this “n”.Nevertheless, no matter which nucleotide is used to replace the “n” atposition 376, the sequence did not give a consensus sequence forsplicing (ag/gtaag etc.). Considering the features of the information ofthe gss database, it was presumed that the sequence [an/gcaag] aroundthe “n” at position 376 was actually [ag/gtaag]. Determination of thefull-length nucleotide sequence of NR10.1 and NR10.2 revealed that thesetwo genes are connected to a different exon at the object obscuresplicing site through alternative splicing, and the C-terminusthereafter encoded different amino acid sequences. Their primarystructure indicates that NR10.1 may encode a transmembrane typehemopoietin receptor protein consisting of 652 amino acids, and thatNR10.2 may encode a soluble secretion type receptor-like proteinconsisting of 252 amino acids. The structural features of these NR10 areas follows:

First, it is predicted that the sequence from the 1st Met to the 32ndAla in the common extracellular domain of NR10.1 and NR10.2 is thetypical secretion signal sequence. Herein, the 1st Met is presumed to bethe translation initiation site because there exists an in frametermination codon at the (−2) position. Next, a typical ligand-bindingdomain exists in the region from the 43rd Cys to the 53rd Cys or the55th Trp residue. In addition, the 81st and 94th Cys correspond to theCys residue repeat conformation well conserved among other hemopoietinreceptor family. Furthermore, a Pro-rich region (PP-W motif) beginningat the consecutive Pro residues at positions 137 and 138 to the 157thTrp residue is conserved, and residues from the 210th Tyr to 215th Argcorresponds to the YR motif above. A typical WSXWS-box (WS motif; SEQ IDNO:22) is also found at residues from the 224th Trp to 228th Ser.

The open reading frame (ORF) of NR10.2 encodes 24 amino acids from theWSXWS sequence (SEQ ID NO:22) and terminates at the stop codonthereafter. Thus, it encodes a soluble hemopoietin receptor-like proteinwithout a transmembrane region. On the other hand, the ORF of NR10.1contains a typical transmembrane domain of 24 amino acids from the 533rdIle to the 556th Leu residue following the above motifs. In addition,the intracellular domain adjacent to the transmembrane domain containsPro residues at positions 571 and 573, corresponding to the Box-1consensus sequence (PXP motif) well conserved among other hemopoietinreceptors and is considered to be implicated in signal transduction.These features above confirm that the NR10 gene encodes a novelhemopoietin receptor protein.

Example 2 Tissue Distribution Determination and Expression PatternAnalysis of NR10 Gene by RT-PCR

mRNA was detected using the RT-PCR method to analyze the expressiondistribution and the expression patterns of NR10.1 and NR10.2 gene indifferent human organs. Oligonucleotide primers with the followingsequences were synthesized for RT-PCR analysis. NR10-S0 primer was usedas a sense primer (downstream orientation), and NR10.1-A0 and NR10.2-A0primer were used as antisense primers (upstream orientation). Theprimers were synthesized and purified as described in Example 2. WhileNR10-S0 was designed so as to correspond to common sequences of NR10.1and NR10.2, NR10.1-A0 and NR10.2-A0 were designed according to specificsequences of NR10.1 and NR10.2, respectively.

hNR10-S0: (SEQ ID NO: 13) 5′-GCA TTC AGG ACA GTC AAC AGT ACC AGC-3′hNR10.1-A0: (SEQ ID NO: 14) 5′-AGC TGG AAT CCT CAG GGT GGC CAC TGG-3′hNR10.2-A0: (SEQ ID NO: 15) 5′-GCC CAT CAC CAG AGT AGA CAG GAC GGG-3′

The templates used were Human Multiple Tissue cDNA (MTC) Panel I(Clontech #K1420-1), Human MTC Panel II (Clontech #K1421-1), HumanImmune System MTC Panel (Clontech #K1426-1), and Human Fetal MTC Panel(Clontech #K1425-1). PCR was performed using Advantage cDNA PolymeraseMix (Clontech #8417-1) on a thermal cycler (Perkin Elmer Gene Amp PCRSystem 2400). NR10-S0 and NR10.1-A0 were used in pair for the detectionof NR10.1. For the detection of NR10.2, [NR10-S0 and NR10.2-A0] primerset was used. PCR was performed by following condition to amplify thetarget gene: a single cycle of “94° C. for 4 min”, 5 cycles of “94° C.for 20 sec, and 72° C. for 1 min”, 5 cycles of “94° C. for 20 sec, and70° C. for 1 min.”, 25 cycles of “94° C. for 20 sec, and 68° C. for 1min”, a single cycle of 72° C. for 3 min, and termination at 4° C.

As shown in FIG. 9, the result was that constitutive gene expression ofNR10.2 was detected at almost a constant level in all examined humanorgans and tissues derived mRNA. In contrast, as shown in FIG. 8, NR10.1gene expression was detected in restricted tissues or organs, and itsexpression level varied significantly. Performing PCR using human G3PDHprimers under the above condition and detecting the expression of thehouse-keeping gene G3PDH, it was confirmed that the number of mRNAcopies among the template mRNA had been normalized. The expression ofNR10.1 gene was found in organs as follows: in human adult, it wasstrongly expressed in heart, placenta, testis, thymus, and peripheralleukocytes, while weak expression was detected in spleen, bone marrow,prostate, ovary, pancreas, and lung; in human fetus, strong expressionwas detected in skeletal muscle, thymus, heart, and kidney, while weakexpression was detected in lung, liver, and spleen. On the other hand,no expression could be detected in brain, skeletal muscle, kidney, smallintestine, or colon in human adult, nor in fetal brain.

The size of the PCR amplification product was 480 by and 243 by forNR10.1 and NR10.2, respectively, which was consistent with the sizescalculated from the determined nucleotide sequences. Thus, the productswere considered to be products of specific PCR amplification reaction.This was further confirmed by Southern blotting as in the following, andthe possibility of that they were non-specific PCR amplificationproducts was denied.

Due to the fact that a strong expression of NR10.1 gene was mainlydetected in those organs containing immune responsible cells andhematopoietic cells and considering the gene expression distribution ofNR10.1, the possibility that NR10 functions as a novel hemopoietinreceptor was strongly suggested. Additionally, the fact that theexpression was also distributed among cells of the genital system andthe endocrine system as well as in heart suggested that NR10 couldregulate not only the immune system and hematopoietic system but alsodiverse physiological functions in the body as well.

The fact that expression of NR10.2 was detected in all organs indicatesthe possibility that cells constituting the subject organs of theanalysis produce active secretory type protein. It is possible that theexpression of NR10 gene is strictly regulated in particular tissues orcell populations through transcriptional regulation and alternativesplicing that determines the functional specificity of these tissues andcells.

Example 3 Verification of the Specificity of PCR Products by SouthernBlotting

In order to verify the specificity of amplification, the RT-PCRamplified target gene product in Example 2 was subjected to Southernblotting using cDNA fragments specific for NR10.1 and NR10.2,respectively, as a probe. At the same time, the amount of RT-PCR productwas quantitatively detected to assess relative gene expression levelsamong different human organs. The RT-PCR product was electrophoresed onan agarose gel, blotted onto a charged nylon membrane (Hybond N(+),Amersham cat#RPN303B), and subjected to hybridization. cDNA fragments ofNR10.1 and NR10.2 obtained in Example 3 were used as probes specific forrespective genes. Probes were prepared using the Mega Prime Kit(Amersham cat#RPN1607), and labeled with radioisotope, [α-³²P]-dCTP(Amersham cat#AA0005). Hybridization was performed using ExpressHyb-ridization Solution (Clontech #8015-2), and after theprehybridization at 68° C. for 30 min, heat denatured labeled probe wasadded to conduct hybridization at 68° C. for 120 min. After subsequentwash in (1) 1×SSC/0.1% SDS at room temperature for 5 min, (2) 1×SSC/0.1%SDS at 50° C. for 30 min, and (3) 0.1×SSC/0.1% SDS at 50° C. for 30 min,the membrane was exposed to an Imaging Plate (FUJI #BAS-III), and NR10specific signal was detected using the Image Analyzer (FUJIX, BAS-2000II).

Detected results for NR10.1 and NR10.2 are shown in FIGS. 10 and 11,respectively. The amplified product in the previous RT-PCR was verifiedas specific amplification products of respective genes. Furthermore, theresult of quantification of relative expression level among each tissuessupported above-mentioned assessment. The detection method for targetgene expression using RT-PCR and Southern blotting in combination isknown to have extremely high sensitivity as compared to other methodsfor expression analysis. Nevertheless, NR10.1 expression was notdetected in the neuronal system such as adult and fetal brains, in adultdigestive tissues. Moreover, no expression was detected in adultskeletal muscle or kidney, where strong expression was recognized infetus.

Example 4 Northern Blot Analysis of NR10 Gene Expression

Northern blot analysis of NR10 gene expression was performed to examinethe expression pattern of NR10 gene in human organs and human tumor celllines, and to determine the size of NR10 transcripts. In addition, thepossibility of whether splice variants other than NR10.1 or NR10.2existed was examined. Human Multiple Tissue Northern (MTN) Blot(Clontech #7760-1), Human MTN Blot II (Clontech #7759-1), Human MTN BlotIII (Clontech #7767-1), and Human Cancer Cell Line MTN Blot (Clontech#7757-1) were used.

The cDNA fragments obtained by 5′-RACE in Example 1 (5) were used as theprobes. Probes were prepared as described in Example 3, using the MegaPrime Kit, and labeled with [α-³²P]dCTP. Hybridization was performedusing Express Hyb-ridization Solution, and after prehybridization at 65°C. for 30 min heat denatured probes were added to conduct hybridizationat 65° C. for 16 hr. After subsequent wash in (1) 1×SSC/0.1% SDS at roomtemperature for 5 min, (2) 1×SSC/0.1% SDS at 48° C. for 30 min, and (3)0.5×SSC/0.1% SDS at 48° C. for 30 min, the membrane was exposed to anImaging Plate as described above, and an attempt to detect NR10 specificsignal was made using an Image Analyzer.

The method, unexpectedly, failed to detect any signal in any of theexamined human organs. This could be because Northern blotting has asignificantly lower sensitivity than RT-PCR and thus failed to detectmRNA with low expression level.

Example 5 Plaque Screening

The above procedure utilized PCR cloning for obtaining the full-lengthcDNA of NR10 gene. There is always the possibility that a point mutationin the product is introduced by PCR cloning. Thus, in order to reconfirmthe nucleotide sequence of the above cDNA clone, plaque hybridizationwas performed using a lambda phage cDNA library to reisolate the targetgene. Human Placenta cDNA library (Clontech #HL1144X), in which theexpression of NR10 gene was confirmed as a result of NR10 geneexpression analysis by RT-PCR, was used for the plaque screening. ThecDNA fragments obtained by 5′-RACE in Example 1 (5) were used as theprobe, as above. Probes were prepared and labeled as in Example 3, usingthe Mega Prime Kit, and labeled with [α-³²P]dCTP. Hybridization wasperformed using Express Hyb-ridization Solution, and afterprehybridization at 65° C. for 30 min heat denatured probes were addedto conduct hybridization at 65° C. for 16 hr. After subsequent wash in(1) 1×SSC/0.1% SDS at room temperature for 5 min, (2) 1×SSC/0.1% SDS at58° C. for 30 min, and (3) 0.5×SSC/0.1% SDS at 58° C. for 30 min, themembrane was exposed to an X-ray film (Kodak, cat#165-1512) to detectNR10 positive plaques.

As a result, no positive clone was obtained. As described in Example 4,one reason why the cDNA clone couldn't be isolated might be that theexpressed copy numbers of the target gene was too small. To isolate thetarget gene, it is favorable to perform plaque hybridization using alambda phage cDNA library derived from human fetal skeletal muscle,which showed the highest expression level of the gene by RT-PCRanalysis.

Example 6 Ligand Screening (1) Construction of NR10 Chimeric Receptor

A screening system is constructed for searching a ligand, a novelhemopoietin, that can specifically bind to NR10. First, the cDNAsequence encoding the extracellular region of NR10.1 (from the 1st Metto the 238th Glu or 1st Met to the 532nd Glu) was amplified by PCR, andthis DNA fragment is bound in frame to DNA fragments encoding thetransmembrane region and the intracellular region of a known hemopoietinreceptor to prepare a fusion sequence encoding a chimeric receptor. Asdescribed above, there are several candidates for the partner, the knownhemopoietin receptor, and among them, the human TPO receptor (HumanMPL-P) is selected. Specifically, after amplifying the DNA sequenceencoding the intracellular region that includes the transmembrane regionof the human TPO receptor by PCR, this sequence was bound to the cDNAsequence encoding the extracellular region of NR10.1 in frame, and wasinserted into a plasmid vector (pEF-BOS) expressible in mammalian cells.The constructed expression vector was named pEF-NR10/TPO-R. A schematicdiagram of the structure of the constructed NR10/TPO-R chimeric receptoris shown in FIG. 12. Together with an expression vector pSV2bsr (KakenPharmaceutical) containing Blastcidin S resistant gene, the NR10/TPO-Rchimeric receptor-expressing vector was introduced into the growthfactor-dependent cell line Ba/F3, and was forced for expression.Gene-introduced cells were selected by culturing under the coexistenceof 8 μg/ml of Blastcidin S hydrochloride (Kaken Pharmaceutical) andIL-3. By transferring the obtained chimeric receptor-introduced cells toan IL-3-free medium, culturing by adding materials expected to contain atarget ligand, it is possible to conduct screening which makes use ofthe fact that survival/proliferation of the cell is possible only when aligand that specifically binds to NR10 is present.

(2) Preparation of NR10/IgG1-Fc Soluble Fusion Protein

NR10/IgG1-Fc soluble fusion protein was prepared to utilize it forsearching cell membrane-bound type ligands, or to detect soluble ligandsthrough BIAcore (Pharmacia) and West-western blotting. A fusion sequenceencoding the soluble fusion protein was prepared by binding the DNAfragment encoding the extracellular region of NR10.1 (from the 1st Metto the 238th Glu or 1st Met to the 532nd Glu) prepared in Example 6(1)with the DNA fragment encoding the Fc region of human immunoglobulinIgG1 in frame. A schematic diagram of the structure of the solublefusion protein encoding the constructed NR10/IgG1-Fc is shown in FIG.12. This fusion gene fragment was inserted into a plasmid vector(pEF-BOS) expressible in mammalian cells, and the constructed expressionvector was named pEF-NR10/IgG1-Fc. After forcing expression of thispEF-NR10/IgG1-Fc in mammalian cells, and selection of stablegene-introduced cells, the recombinant protein secreted into the culturesupernatant can be purified by immunoprecipitation using anti-humanIgG1-Fc antibody, or by affinity columns, etc.

(3) Construction of an Expression System of NR10.2 and Purification ofthe Recombinant NR10.2 Protein

The recombinant NR10.2 protein was prepared to utilize it for searchingcell membrane-bound ligands, or the detection of soluble ligands usingBIAcore (Pharmacia) or West-western-blotting. The stop codon of theamino acid coding sequence of NR10.2 cDNA was replaced by point mutationto a nucleotide sequence encoding an arbitrary amino acid residue, andthen, was bound to the nucleotide sequence encoding the FLAG peptide inframe. This bound fragment was inserted into a plasmid vectorexpressible within mammalian cells, and the constructed expressionvector was named pEF-BOS/NR10.2 FLAG. FIG. 12 shows a schematic diagramof the structure of the insert NR10.2 FLAG within the constructedexpression vector. After forced-expression of this pEF-BOS/NR10.2 FLAGin mammalian cells and selection of stable gene-introduced cells, therecombinant protein secreted into the culture supernatant can beimmunoprecipitated using anti-FLAG peptide antibody, or may be purifiedby affinity columns, etc.

Example 7 Isolation of NR10.3 Gene (1) Design of Oligonucleotide Primers

Isolation of NR10.1 gene was conducted again to obtain the cDNAcomprising a continuous full-length coding sequence. First, 5′-UTR and3′-UTR within the nucleotide sequence of NR10.1 cDNA was selected todesign sense and antisense primers (downstream and upstream orientation,respectively) with sequences as follows. Primers were synthesized as inExample 1 (2) on an ABI 394 DNA/RNA Synthesizer under the conditionwhere a trityl group was attached to the 5′-terminus. The product waspurified using an OPC column (ABI #400771) to obtain full-lengthprimers.

NR10-5UTR (SN); (SEQ ID NO: 18) 5′-CCC CTG ATA CAT GAA GCT CTC TCC CCAGCC-3′ NR10-3UTR (AS); (SEQ ID NO: 19) 5′-CCA GTC TTC GGA GAT GGT TCTCTT GGG GCC-3′

(2) PCR Cloning

In order to isolate the full length CDS of NR10, PCR cloning wasperformed using NR10-5UTR and NR10-3UTR primers as sense and antisenseprimers, respectively. Human Placenta Marathon-Ready cDNA Library(Clontech #7411-1) was used as the template. PCR experiment wasperformed using the Advantage cDNA Polymerase Mix (Clontech #8417-1) ona thermal cycler Perkin Elmer Gene Amp PCR System 2400. PCR wasperformed by a single cycle of “94° C. for 4 min”, 5 cycles of “94° C.for 20 sec, and 72° C. for 90 sec”, 5 cycles of “94° C. for 20 sec, and70° C. for 90 sec”, 28 cycles of “94° C. for 20 sec, and 68° C. for 90sec”, a single cycle of 72° C. for 3 min, and was terminated at 4° C. Asa result, an amplification product of 2119 by was obtained.

The obtained PCR product was subcloned into pGEM-T Easy vector (Promega#A1360) as in Example 1 (3), and the nucleotide sequence was determined.Recombination of the PCR product into the pGEM-T Easy vector wasperformed using T4 DNA Ligase (Promega #A1360) in a reaction of 12 hrsat 4° C. The recombinant of the PCR product and the pGEM-T Easy vectorwas obtained by transformation of DH5 alpha E. coli (Toyobo#DNA-903),and Insert Check Ready Blue (TOYOBO #PIK-201) was used for theselection. The nucleotide sequence was determined using the BigDyeTerminator Cycle Sequencing SF Ready Reaction Kit (ABI/Perkin Elmer#4303150) and the ABI PRISM 377 DNA Sequencer. The nucleotide sequencesof the whole insert fragments from 5 independent clones of therecombinant were determined. As a result, the nucleotide sequence of acDNA clone that may encode the full length CDS of NR10 including thetransmembrane region was determined. However, the determined sequencewas not recognized as that of NR10.1, but instead it was a cDNA clonewhich could encode a transmembrane type of receptor protein of 662 aminoacids. The clone was named NR10.3 so as to distinguish it from theNR10.1.

E. coli containing this cDNA clone was deposited in National Instituteof Bioscience and Human-Technology, Agency of Industrial Science andTechnology.

Depositary institution: National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry.

Address: 1-1-3 Higashi, Tsukuba, Ibaraki 305-8566, Japan.

Deposition date (original date): Jul. 23, 1999 (Heisei 11).

Accession No. Seimeiken Jyouki Dai 6793 Go (FERM BP-6793).

As compared with NR10.1, the NR10.3 cDNA clone has a single nucleotidedeletion in the adenine cluster at the proximity of the stop codonleading to a frame shift. Thereby, NR10.1 and NR10.3 exhibit differencein the reading frame of the amino acid sequence proximal to the stopcodon. The decided nucleotide sequence of NR10.3 and the amino acidsequence encoded by it are shown in SEQ ID NOs:16 and 17, respectively,as well as in FIGS. 13 and 14.

(3) Significance of the existence of NR10.1 and NR10.3

As described above, the difference between NR10.1 and NR10.3 is causedby the difference of a single nucleotide at a position near the stopcodon, and not by different transcription products due to splicingmutants. Since NR10.1 and NR10.3 cDNA clone are identical except for thedeletion of the single nucleotide, the hematopoietic factor receptorproteins encoded by them are presumed to be functionally equivalent.However, such single nucleotide deletion or point mutation could play arole in certain disease, or the sequence diversity may be caused familyor race dependently.

Example 8 Chromosomal Location of the NR10 (1) Design of OligonucleotidePrimers

In order to construct a chromosome map of NR10, an oligonucleotideprimer, NR10-intron, with the following sequence was synthesized.NR10-intron primer was designed as a sense primer (downstreamorientation) by selecting the sequence of an intron site, nottranscribed into NR10 mRNA, within the sequence of AQ022781 deposited inthe gss database. The primer was synthesized as described in Example 1(2) using an ABI 394 DNA/RNA Synthesizer under condition where a tritylgroup is attached to the 5′-terminus, and purified on an OPC column (ABI#400771) to obtain a full-length product.

NR10-intron (SN): (SEQ ID NO: 20) 5′-CTG TGT AAG TAC CAA TTG TTC CCAGGC-3′

(2) Chromosome Mapping of the NR10 Gene

In order to make a chromosome map of NR10, PCR analysis was performedusing respective DNA obtained from human/mouse somatic cell systemhaving 24 chromosomes (Dubois et al., Genomics, 16:315-319, 1993).

NR10-intron primer of Example 8 (1) and NA10-A1 primer produced inExample 1 (2) were used as sense and antisense primers, respectively.PCR experiment was performed using Advantage cDNA Polymerase Mix(Clonetech #8417-1) on a thermal cycler Perkin Elmer Gene Amp PCR System2400 under the following PCR condition. As a result, a 359 byamplification product was amplified, which suggested the existence ofNR10 gene on human chromosome 5.

PCR was performed by a single cycle of “94° C. for 4 min”, 5 cycles of“94° C. for 20 sec, and 70° C. for 60 sec”, 28 cycles of “94° C. for 20sec, and 68° C. for 60 sec”, and a single cycle of 72° C. for 3 min, andwas terminated at 4° C.

The obtained PCR product was cloned into pGEM-T Easy vector (Promega#A1360) as described in Example 1 (3), and the nucleotide sequence wasdetermined using an ABI PRISM 337 DNA Sequencer. Analysis of thenucleotide sequence of the whole insert fragment from eight independentrecombinant clones confirmed that the PCR product had the nucleotidesequence of the target genomic DNA fragment containing a partialsequence of NR10, and not a product due to non-specific amplification.

The above result also confirmed that the primer set was working in aspecific manner. Subsequently, the locus of the NR10 gene was determinedusing the GeneBridge 4 radiation hybrid panel 93 (Walter et al., NatureGenetics, 7:22-28, 1994). PCR analysis was performed using theGeneBridge 4 radiation hybrid panel 93 as a template and N10-intron andNR10-A1 primers under the same condition as above. The amount ofamplified products from respective hybrids were quantitatively assessedas plus or minus, and the result was converted to binary code. Using theprogram in the server at the website of carbon.wi.mit.edu:8000/cgi-bin/contig/rhmapper.pl, the result was compared with similarcodes of gene map marker genes used for constructing frame-work maps,and the location on the chromosome was determined. As a result, NR10 wasmapped on chromosome 5 proximal to the centrosome, and was furtherconfirmed that it exists between the markers WI-3071 (60-61 cM) andAFM183YB8 (67 cM).

Human gp130 and LIF receptor genes, which were used in the originaldatabase search by the inventors, were also mapped on regions ofchromosome 5. More specifically, the human gp130 gene was mapped onchromosome 5 q11 (67.2-69.6 cM), and human LIF receptor gene was mappedon chromosome 5 p12-p13 (59.9-61.1 cM).

From the point of evolutionary genetics, it is also of great importancethat the NR10 gene was mapped to the region 61-67 cM on chromosome 5, aregion between the two genes. That is, the three genes, human gp130,human LIF receptor, and human NR10 genes, of the same receptor family,whose structures show relatively high similarity in the family, arelocated close to each other in an extremely restricted region of thesame human chromosome 5. This fact supports the theory that the threedifferent receptor genes are derived from a same ancestral gene, andthat they went through genetical evolution during the long history ofbiological evolution to achieve diversity not only in their structurebut also functions.

INDUSTRIAL APPLICABILITY

The present invention provides novel hemopoietin receptor proteins andDNA encoding same. The present invention also provides: a vector intowhich the DNA has been inserted, a transformant harboring the DNA, and amethod for producing recombinant proteins using the transformant. Itfurther provides a method of screening for a compound or a naturalligand that binds to the protein. The protein of the invention isthought to be associated with immunological and hematopoietic functions.Therefore, it is expected that the proteins of this invention can beapplied for diagnosis and treatment of diseases related with immunityand hematopoiesis.

As described above, the NR10 gene is expected to provide a useful sourcefor obtaining novel hematopoietic factors or agonists that are capableof functionally binding to the receptor protein encoded by the gene. Itis expected that cellular immunity or hematopoietic function in vivowill be enhanced by administering such functional binding substances orspecific antibodies that can activate the function of NR10 molecule tothe organism. Thus, it is possible to develop a drug for clinicalapplication that promotes proliferation or differentiation of the immuneresponsible cells or hematopoietic cells, or that activates the functionof the immune cells by using the NR10 gene. It is also possible to usesuch drugs to enhance the cytotoxic immunity against particular types oftumor. It is possible that NR10.1 is expressed in a restrictedpopulation of cells in the hematopoietic tissues. Accordingly, anti-NR10antibodies would be useful for the isolation of such cell populations,which may be used for cell transplantation treatments.

On the other hand, NR10.2, a splice variant of NR10, may be used as aninhibitor for the NR10 ligand, as a decoy type receptor. Further, it isexpected that by administering antagonists that can bind, functionallyto the NR10 molecule, or other inhibitors, as well as specificantibodies that can inhibit the molecular function of NR10 to theorganism, it is possible to suppress the cellular immunity or inhibitthe proliferation of hematopoietic cells in vivo. Thus, it is possibleto apply such inhibitors to the development of a drug for clinicalapplication that inhibits the proliferation or differentiation of theimmune responsible cells or hematopoietic cells, or suppresses theimmune function or inflammation. Specifically, it is possible to usesuch inhibitors to suppress the onset of autoimmune diseases arisingfrom autoimmunity, or tissue rejection by the immune system of theliving body, the primary problem in transplantation. Furthermore, theinhibitors may be effectively used to treat such diseases caused by theabnormally upregulated immune response. Thus, it is possible to use theinhibitors to treat a variety of allergies that are specific toparticular antigens, such as metal and pollen.

1-19. (canceled)
 20. An isolated nucleic acid comprising a nucleotidesequence encoding a protein selected from the group consisting of: (a) aprotein comprising the amino acid sequence from Ala at position 33 toGlu at position 532 of SEQ ID NO:2; (b) a protein comprising the aminoacid sequence from Ala at position 33 to Glu at position 532 of SEQ IDNO:2 in which one to 30 amino acids are deleted, added and/orsubstituted, wherein said protein has a ligand-binding domain in theregion corresponding to the Cys at position 43 to the Cys at position 53of SEQ ID NO:2; and (c) a protein comprising the amino acid sequencefrom Ala at position 33 to Val at position 252 of SEQ ID NO:4 in whichone to 30 amino acids are deleted, added and/or substituted, whereinsaid protein has a ligand-binding domain in the region corresponding tothe Cys at position 43 to the Cys at position 53 of SEQ ID NO:4.
 21. Avector into which the nucleic acid of claim 20 is inserted.
 22. Anisolated cell harboring the nucleic acid of claim
 20. 23. An isolatedcell harboring the vector of claim
 21. 24. A method for producing apolypeptide, the method comprising the steps of culturing the cell ofclaim 22 and recovering the protein from the cell or the culturesupernatant thereof.
 25. A method for producing a polypeptide, themethod comprising the steps of culturing the cell of claim 23 andrecovering the protein from the cell or the culture supernatant thereof.26. The isolated nucleic acid of claim 20, wherein the nucleic acidencodes a protein comprising the amino acid sequence from Ala atposition 33 to Glu at position 532 of SEQ ID NO:2.
 27. The isolatednucleic acid of claim 20, wherein the nucleic acid encodes a proteincomprising the amino acid sequence from Ala at position 33 to Glu atposition 532 of SEQ ID NO:2 in which one to 30 amino acids are deleted,added and/or substituted, wherein said protein has a ligand-bindingdomain in the region corresponding to the Cys at position 43 to the Cysat position 53 of SEQ ID NO:2.
 28. The isolated nucleic acid of claim20, wherein the nucleic acid encodes a protein comprising the amino acidsequence from Ala at position 33 to Val at position 252 of SEQ ID NO:4in which one to 30 amino acids are deleted, added and/or substituted,wherein said protein has a ligand-binding domain in the regioncorresponding to the Cys at position 43 to the Cys at position 53 of SEQID NO:4.
 29. The isolated nucleic acid of claim 20, wherein the nucleicacid encodes a protein consisting of the amino acid sequence from Ala atposition 33 to Glu at position 532 of SEQ ID NO:2.
 30. The isolatednucleic acid of claim 20, wherein the nucleic acid encodes a proteinconsisting of the amino acid sequence from Ala at position 33 to Glu atposition 532 of SEQ ID NO:2 in which one to 30 amino acids are deleted,added and/or substituted, wherein said protein has a ligand-bindingdomain in the region corresponding to the Cys at position 43 to the Cysat position 53 of SEQ ID NO:2.
 31. The isolated nucleic acid of claim20, wherein the nucleic acid encodes a protein consisting of the aminoacid sequence from Ala at position 33 to Asp at position 652 of SEQ IDNO:2 in which one to 30 amino acids are deleted, added and/orsubstituted, wherein said protein has a ligand-binding domain in theregion corresponding to the Cys at position 43 to the Cys at position 53of SEQ ID NO:2.
 32. The isolated nucleic acid of claim 20, wherein thenucleic acid encodes a protein consisting of the amino acid sequencefrom Ala at position 33 to Val at position 252 of SEQ ID NO:4 in whichone to 30 amino acids are deleted, added and/or substituted, whereinsaid protein has a ligand-binding domain in the region corresponding tothe Cys at position 43 to the Cys at position 53 of SEQ ID NO:4.
 33. Thenucleic acid of claim 20, wherein the nucleic acid comprises anucleotide sequence encoding a protein comprising the amino acidsequence from Ala at position 33 to Ile at position 662 of SEQ ID NO:17in which one to 30 amino acids are deleted, added and/or substituted,wherein said protein has a ligand binding domain in the regioncorresponding to the Cys at position 43 to the Cys at position 53 of SEQID NO:17.
 34. The nucleic acid of claim 33, wherein the nucleotidesequence encodes a protein consisting of the amino acid sequence fromAla at position 33 to Ile at position 662 of SEQ ID NO:17 in which oneto 30 amino acids are deleted, added and/or substituted, wherein saidprotein has a ligand binding domain in the region corresponding to theCys at position 43 to the Cys at position 53 of SEQ ID NO:17.
 35. Avector into which the nucleic acid of claim 33 is inserted.
 36. Anisolated cell harboring the nucleic acid of claim
 33. 37. An isolatedcell harboring the vector of claim
 35. 38. A method for producing apolypeptide, the method comprising the steps of culturing the cell ofclaim 36 and recovering the protein from the cell or the culturesupernatant thereof.
 39. A method for producing a polypeptide, themethod comprising the steps of culturing the cell of claim 37 andrecovering the protein from the cell or the culture supernatant thereof.